Human Reproduction Vol.20, No.12 pp. 3469–3475, 2005
doi:10.1093/humrep/dei247
Advance Access publication August 25, 2005.
Flow cytometry of human semen: a preliminary study of a
non-invasive method for the detection of spermatogenetic
defects
N.Levek-Motola1, Y.Soffer2, L.Shochat1, A.Raziel2, L.M.Lewin1 and R.Golan1,3
1
Department of Clinical Biochemistry, Sackler Medical School, Tel Aviv University, Ramat Aviv and 2Male Infertility Unit, Assaf
HaRofe Medical Center, Zerifin, Israel
3
Corresponding author. E-mail: [email protected]
BACKGROUND: The pathway of spermatogenesis involves the conversion of diploid stem cells (spermatogonia) to
tetraploid primary spermatocytes, followed by meiosis and two cell divisions, first forming diploid secondary spermatocytes and then haploid round spermatids. Differentiation of round spermatids results in spermatozoa containing
condensed chromatin. It has long been known that semen from patients with non-obstructive azoospermia or oligospermia contains small numbers of immature germinal cells. In this article, a flow cytometric procedure is
described for assessing defects in spermatogenesis by identifying the ploidy of those immature cells. METHODS:
Cells in semen samples from 44 infertile patients and 14 controls were stained with propidium iodide, which displays
red fluorescence when intercalated between bases in double-stranded DNA. The resulting cell suspension was examined by quantitative flow cytometry, with excitation by laser light (488 nm) and red fluorescence recorded on a logarithmic scale to allow easy differentiation between intensities of tetraploid, diploid and haploid round spermatids,
and spermatozoa containing condensed chromatin. RESULTS: The flow cytometric method differentiated between
cases of ‘Sertoli cell-only’ syndrome (complete absence of tetraploid and haploid cells) and cases where spermatogenesis was blocked in meiosis or in spermiogenesis. Flow cytometric histograms from semen samples from normozoospermic, oligozoospermic and azoospermic patients fell into patterns that correlated well with the results obtained
from testis histology findings. CONCLUSIONS: The method described may serve as a simple, non-invasive and reliable assay to help clinicians counsel patients with severe male infertility before referring them for testicular surgery to
locate spermatozoa for ICSI.
Key words: flow cytometry/male infertility/non-obstructive azoospermia/spermatogenesis
Introduction
Spermatogenesis, which takes place in the seminiferous
tubules of the testis, consists of a cascade of cytological, morphological and biochemical transformations. The diploid stem
cells (spermatogonia) either undergo mitotic divisions to reproduce themselves or differentiate into primary spermatocytes
(4N, containing twice as much DNA per cell). These cells
undergo meiosis, during which crossing over of DNA occurs,
followed by two cell divisions to produce four round haploid
cells (1N, spermatids). In the next process, spermiogenesis,
morphological changes result in elongation of the spermatids,
condensation of nuclear chromatin and production of flagella.
In male infertility, the spermatogenesis pathway is generally
disturbed, leading to abnormal production of spermatozoa with
impaired morphology (teratozoospermia), decreased motility
(asthenozoospermia) and reduced production of mature spermatozoa ranging from mild oligozoospermia to azoospermia.
Semen analysis according to World Health Organization
(1999) criteria allows evaluation of the results of this disturbed
process but not of its nature. More sophisticated means are
needed to reveal the underlying spermatogenic defect. Histological examination of testicular biopsy samples is suitable for
this purpose, but it is invasive and therefore is not generally
indicated in routine clinical practice.
In previous investigations from this laboratory, flow cytometric ploidy analysis of testis single cell suspensions stained
with propidium iodide (PI) was used to study spermatogenesis
in the hamster (Golan et al., 2000). Differences in flow cytometric histograms between control cell suspensions and those
from cryptorchid testes (Vigodner et al., 2003) or from animals
treated with procarbazine (Weissenberg et al., 2002) demonstrated inhibition at various loci of the spermatogenesis pathway. Similar studies done on testes of rats following
cyclophosphamide and ethynylestradiol administration (Katoh
et al., 2002) or doxorubacin (Suter et al., 1997) yielded the
same type of results. In humans, flow cytometry has been performed on testicular material and has been found to be valuable
(Giwercman et al., 1994; Coskun et al., 2002). Kostakapoulos
© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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3469
N.Levek-Motola et al.
et al. (2002) and Dey et al. (2000) performed flow cytometry
on testicular material for DNA ploidy determination. In these
studies, comparison with testicular cytopathology found flow
cytometry of testis cells to be highly valuable. Flow cytometry
was not performed on semen in these studies.
In the present investigation, we found semen flow cytometry
(SFC), a non-invasive method, to be an efficient means for
detecting defects in human spermatogenesis. We used PI to
stain double-stranded DNA in the cells present in the semen of
normal and azoospermic patients. These cells were subjected
to flow cytometry for quantitative estimation of cells containing chromatin with fluorescence characteristic of tetraploid
(4N), diploid (2N), haploid (1N) and other cells (less fluorescent than the 1N samples). This allowed us to assess the presence of primary spermatocytes and of spermatids containing
uncondensed, condensing and fully condensed chromatin.
Comparison of these results with those from histological examination of testicular biopsies of the same patients was used to
validate the conclusions derived from analysis of the flow
cytometric data.
Materials and methods
Source and handling of samples
Semen samples remaining after routine semen analysis (n = 58) from
the male infertility clinic at Assaf HaRofe Medical Center were
examined.
The study group comprised 44 samples from infertile men and 14
from men with normal semen parameters (World Health Organization, 1999) as controls. Semen characteristics of the normal control
group were as follows: volume range 1.5–6 ml, median 2.8 ml; sperm
count range 36–179 × 106/ml, median 104 × 106/ml; motility range
40–60%, median 50%; motility grade (on a scale from 0 to 4) range
3.1–3.5, median 3.4; normal forms (strict criteria) range 6–11%,
median 7%.
Of the 44 samples from infertile men, 10 were from oligo-teratoasthenozoospermic patients (OTA): six cases with extreme OTA, i.e.
few spermatozoa found only after centrifugation and careful examination of the pellet in small drops under an inverted microscope (Ron-El
et al., 1997); and four with OTA (in this study defined as <12 × 106 in
total sperm count per ejaculate, <25% motility and <4% normal
forms). There was one case with severe hypomotility (<10% motility
with normal sperm count) and 33 samples with azoospermia.
Azoospermia was assessed in repeated semen examinations when no
spermatozoa were found following centrifugation and careful examination of the pellet (see above). Thirty cases were diagnosed as nonobstuctive azoospermia (NOA) according to clinical data (FSH value
>12 IU/l and low testis volume <12 ml each) and/or testicular biopsy
findings (hypospermatogenesis, maturation arrest or Sertoli cell-only
syndrome). Two cases were diagnosed as hypogonadotrophic hypogonadism (FSH <0.5 IU/l, very small testes and eunichoidism). One case
of azoospermia was obstructive, in a previously fertile man, following
voluntary vasectomy.
In all the 44 cases, qualitative SFC analysis was done, and in 33 of
these quantitative analysis was also performed, as a high enough
semen volume was available for this procedure.
The samples from infertile men were divided into two portions.
One was centrifuged to concentrate the sample for qualitative analysis. The other, used for quantitative analysis, was diluted 1:2 in TNE
buffer [Tris-hydroxymethylaminomethane (Merck A 943) 0.01 mol/l,
NaCl (Biolab 69684) 0.15 mol/, EDTA (Merck A8382) 1 mmol/l,
3470
pH 7.4] supplemented with glycerol (J.T.Baker 2136-01) (10% v/v)
and frozen at –20°C for storage until analysis.
Qualitative analysis of semen samples using flow cytometry
Qualitative analysis using flow cytometry was performed on the
semen samples. To 200 μl of pathological samples or 50 μl of the
normal samples diluted with 150 μl of TNE buffer, were added 25 μl
of Igepal (Sigma, I 3021) (octyl phenoxy) polyethoxyethanol
(0.25% final concentration) in bovine serum albumin (BSA) (0.1%
final concentration) and 25 μl of RNase type IIa (Sigma, R5000)
(0.25 μg/ml final concentration) (Sigma, A 7906). Samples were
then incubated for 15 min at 33°C before placing the reaction mixture in an ice bath. PI (Sigma, P 4170) (200 μl final concentration 25
μg/ml) and 5 μl of fluorescent beads (Molecular Probes, FluoSphere) (106/ml)were added and the samples were aspirated into a
flow cytometer (Becton Dickinson FACSort Flow Cytometer, San
Jose, CA). Red fluorescence (BP 650 LP filter) emitted from individual cells was recorded from ∼10 000 cells per sample after excitation with a 488 nm argon laser using a logarithmic scale to allow all
cells from haploid to tetraploid to appear as peaks in the resulting
histograms. Before each experiment, the flow cytometer was calibrated by setting the peak for the fluorescent beads at a value of 186.
The diploid peak was then found at a value of 24.5. Results were
analysed using the WINMDI data processing program (J.Trotter,
http://facs.scripps.edu) and displayed as histograms showing red fluorescence versus the number of cells. Staining of DNA with PI
allowed us to distinguish between four different cell populations
according to their relative ploidy: tetraploid (4N), diploid (2N), haploid immature cells (round and elongating spermatids 1N) and haploid mature (elongated spermatids 1N) cells composed of spermatids
containing condensed chromatin.
For quantitative estimation of cell populations, the above staining
procedure was used, with a measured amount of fluorescent beads
(50 μl of 4 × 106/ml) added as internal standard in each reaction mixture. Samples containing known amounts of spermatozoa were treated
as described above and subjected to flow cytometry. The number of
cells counted per 6000 beads was used to prepare a linear standard
curve (Figure 1). Semen samples were treated in the same way. Using
the WINMDI program, regions conforming to 4N, 2N and 1N were
drawn and events were counted with reference to 6000 beads. The
results were calculated according to the linear curve, and the number
of cells was calculated by using the formula:
C=
Y×X
V
where C = numbers of cells in each population; Y = numbers of cells
calculated from the linear curve; X = dilution factor; and V = volume
of sample.
Testicular biopsy and histology
Bilateral testicular biopsy was performed on azoospermic patients
only if spermatozoa were not found in the semen sample following a
careful semen search (Ron-El et al., 1997) on the day of the scheduled
biopsy. Testicular biopsy was performed through two small incisions
(<0.5 cm) in each testis. Testicular material was minced, dispersed in
the appropriate medium, incubated for at least 2 h and observed under
×400 magnification using a phase contrast microscope. If motile
sperm were found, ICSI was performed, and all remaining motile
sperm were cryopreserved for future use. The remaining testicular
Flow cytometry of human semen
Pattern 2 (Figure 2C)
Cells in ml sample
250000
Diploid and tetraploid cells without haploid cells. Quantification
(Table II) showed a mean value of only 0.17 × 106 tetraploid
cells/ml. This may suggest maturation arrest which allowed
production of only a few primary spermatocytes but did not
permit further differentiation to spermatids.
200000
150000
100000
50000
0
0
1000
2000
3000
4000
5000
6000
Cells per 6000 beads
Figure 1. Typical standard curve. Cells were stained with propidium
iodide and subjected to flow cytometry as described in Materials and
methods. The x-axis represents the number of cells counted per 6000
beads. The y-axis represents the number of cells per ml in the measured
sample.
material was routinely fixed in Stieve solution and sent to the Pathology
Laboratory. Samples were embedded in paraffin, sectioned and
stained with haematoxylin–eosin. Slides were observed under ×400
magnification.
Validation of the SFC method
Seventeen cases with testicular histology were available for validation
of the SFC method. All cases were qualitatively examined by SFC. In
five cases, a quantitative analysis could also be done (see above). Histology findings were correlated with the SFC results.
Results
Data from flow cytometric analyses are presented in the
form of histograms in which the x-axis represents the intensity of fluorescence on a logarithmic scale and the y-axis the
number of cells with the corresponding fluorescence intensity. In histograms from normal fertile control semen samples, a single peak of mature haploid cells was observed
(Figure 2A). Quantitative data demonstrated the presence of
∼10 8spermatozoa/ml comprising >99% of the cells present.
Five patterns were seen in histograms of semen from infertile patients.
Pattern 1 (Figure 2B)
Diploid cells with the complete absence of haploid and tetraploid cells. Very few cells were present, as revealed by quantitative analysis (Table I) which showed a mean value of 0.21
× 106cells/ml. A semen sample from a patient with obstructive azoospermia (subject 5) contained 0.33 × 106 diploid
cells that were therefore not of testicular origin. These cells
may have included leukocytes and somatic cells contributed
to semen from various organs of the reproductive tract. Such
cells could also be present in normal semen and in semen
from all classes of infertile males. Our SFC method could not
separate these diploid cells into subgroups and, therefore, the
presence of ‘diploid cells’ in these semen samples could not
help us further assess the aetiology of azoospermia. This
group included five cases with NOA, one case with hypogonadotrophic hypogonadism and one with obstructive
azoospermia (Table I).
Pattern 3 (Figure 2D, Table III)
Diploid, tetraploid and haploid cells with different degrees of
chromatin condensation were seen but no condensed haploid
cells were present. Five haploid cell types were defined according to their fluorescence intensity (FI): HC = condensed
FI <4.9, haploid non-condensed types HNC1 FI 13–14, HNC2
FI 11–12.9, HNC3 FI 8–10.9 and HNC4 FI 4.5–7.9, where the
diploid fluorescence peak was 24.5. These cases might indicate
a partial arrest of meiosis (resulting in an accumulation of
tetraploid cells) and blocks in spermiogenesis since no mature
spermatozoa could be demonstrated.
Pattern 4 (Figure 2E, Table IV)
Diploid and uncondensed haploid cells without condensed
haploid cells. This might suggest the presence of two inhibitory sites in spermatogenesis, a partial block before the
production of primary spermatocytes and another in spermiogenesis.
Pattern 5 (Figure 2F, Table V)
Diploid, tetraploid and haploid cells with various degrees
of condensation including mature haploid cells. This group
of hypospermatogenesis demonstrated various degrees of
inhibition of chromatin condensation during spermiogenesis.
Validation of SFC as a reliable means of assessing spermatogenesis in fertile and infertile men was done by comparing
SFC results with those from testicular histology:
In three cases belonging to pattern 1 and included in this validation study, using SFC, only diploid cells were demonstrated,
and histology showed Sertoli cell-only syndrome with very
few spermatogonia in one of the samples.
In 11 cases belonging to pattern 2, using SFC, diploid and
tetraploid cells were demonstrated while histology demonstrated maturation arrest in five cases, Sertoli cell-only
syndrome in five cases and very few mature spermatozoa in
one case.
In three cases belonging to pattern 3, SFC detected diploid,
tetraploid and HNC cells, while histology demonstrated one
case with Sertoli cell-only syndrome, one with maturation
arrest at the stage of primary spermatocyte and one with a few
round spermatids.
No biopsy was done in our cases belonging to patterns 4 and 5
since haploid cells were present in the semen and used in ICSI.
To summarize: in nine out of 17 cases, a good concordance
was found between SFC and testicular histology. In seven
cases, SFC was able to detect additional cell populations not
seen by histology. In only one case did histology find mature
spermatozoa not detected by SFC.
3471
N.Levek-Motola et al.
Figure 2. Representative flow cytometry histograms obtained by propidium iodide staining of semen samples. (A) Normal sample; (B) pattern 1;
(C) pattern 2; (D) pattern 3; (E) pattern 4; (F) pattern 5. The dashed line represents the peak of normal spermatozoa. The x-axis represents the
intensity of red fluorescence (FL2-H, a 3 decade logarithmic scale). The y-axis represents the numbers of cells per channel on a linear scale.
T = tetraploid cells; D = diploid cells; HNC = haploid non-condensed; and HC = haploid condensed.
Discussion
It has long been known that semen from azoospermic and
oligozoospermic men contains immature cells shed from the
testis. The purpose of this research has been to test whether
analysis of these cells provides a useful non-invasive method
for assessing defects in spermatogenesis. Hacker-Klom et al.
3472
(1999) used flow cytometry to demonstrate the presence of
various kinds of cells in semen. They concluded that SFC is a
fast method, which adds valuable information to that obtained by
routine semen analysis. However, the relationship between
various seminal cell distribution patterns and the arrest of
development at various stages of spermatogenesis was not
Flow cytometry of human semen
Table I. Quantitative estimation of cells in semen from azoospermic samples where flow cytometric histograms fell into pattern1
H total (106 cells/ml)
HC (106 cells/ml)
HNC (106 cells/ml)
D (106 cells/ml)
T (106 cells/ml)
Sample
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.01
0.05
0.19
0.22
0.33
0.33
0.37
0.01–0.37
0.21 ± 0.14
–
–
–
–
–
–
–
–
1a
2b
3a
4a
5c
6a
7a
Range
Mean ± SD
T = tetraploid cells; D = diploid cells; HNC = haploid non-condensed; and HC = haploid condensed.
Non-obstructive azoospermia.
Hypogonadotrophic hypogonadism.
c
Obstructive azoospermia.
a
b
Table II. Quantitative estimation of cells in semen from azoospermic samples where flow cytometric histograms fell into pattern 2
HC (106 cells/ml)
HNC (106 cells/ml)
D (106 cells/ml
T (106 cells/ml)
Samples
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.18
0.02
0.29
0.02
0.48
5.40
0.02–5.4
1.5 ± 2.3
<0.01
0.02
0.03
0.04
0.06
0.44
0.01–0.44
0.17 ± 0.2
1
2
3
4
5
6
Range
Mean ± SD
T = tetraploid cells; D = diploid cells; HNC = haploid non-condensed; and HC = haploid condensed. All cases were non-obstructive azoospermia.
Table III. Quantitative estimation of cells in semen from azoospermic samples where flow cytometric histograms fell into pattern 3
H total (106 cells/ml)
0.01
0.05
<0.01
0.05
0.16
3.4
0.19
2.8
3.4
0.83
1.74
0.01–3.4
1.15 ± 1.42
HC (106 cells/ml)
–
–
–
–
–
–
–
–
–
–
–
–
–
HNC (106 cells/ml)
HNC4
HNC3
HNC2
HNC1
0.01
–
–
–
–
–
–
1.30
–
0.18
0.79
0–1.30
0.57 ± 0.59
0.05
–
–
–
2.8
–
–
3.4
–
–
0.05–3.4
–
–
<0.01
0.05
0.16
0.60
0.19
1.50
–
0.65
0.95
0–1.5
0.56 ± 0.5
–
–
–
–
–
–
–
–
–
–
–
–
–
D (106 cells/ml)
T (106 cells/ml
Samples
0.05
0.05
0.12
0.04
0.96
0.41
0.53
0.51
0.89
0.95
2.00
0.04–2.00
0.78 ± 0.58
<0.01a
<0.01a
0.03
0.06
0.09
0.12
0.15
0.20
0.28
0.45
1.00
0.01–1.00
0.26 ± 0.30
1b
2a
3a
4c
5a
6c
7c
8a
9a
10a
11c
range
Mean±S.d
T = tetraploid cells; D = diploid cells; HNC = haploid noncondensed types; HNC1 = fluorescence intensity (FI) 13–14; HNC2 = FI 11–12.9; HNC3 = FI 8–10.9;
HNC4 = FI 4.5–7.9, where the diploid fluorescence peak was 24.5; and HC = haploid condensed.
Non-obstructive azoospermia.
b
Hypogonadotrophic hypogonadism.
c
Extreme oligo-terato-asthenozoospermia.
a
analysed. Our study adds this important information. Fossa
et al. (1989) performed SFC analysis of cell ploidy in a group
of unilaterally orchiectomized patients with cancer and found it
useful in assessing spermatogenesis in these patients. Other
studies using a different technique of flow cytometry for
analysis of defects in sperm chromatin in human semen have
been conducted by Evenson et al. (1991). SFC was also used in
the analysis of various defects of sperm for the assement of
sperm quality and fertility potential (Graham, 2001; Gillan
et al., 2005).
In our study of semen from normospermic samples, 96.7%
(mean value) of cells were found to be mature haploid, 2.4%
haploids with uncondensed chromatin, 0.7% diploid cells and
only traces of tetraploid cells (0.2%). These results are in
agreement with those of Hacker-Klom et al. (1999). Diploid
cells were found in all semen samples, from either germinal or
3473
N.Levek-Motola et al.
Table IV. Quantitative estimation of cells in semen from azoospermic samples where flow cytometric histograms fell into pattern 4
H total (106 cells/ml)
3.6
0.08
0.86
0.083.6
1.51 ± 1.84
HC (106 cells/ml)
–
–
–
–
–
HNC (106 cells/ml)
HNC4
HNC3
HNC2
HNC1
3.6
0.08
–
–
–
–
–
0.86
–
–
–
–
–
–
–
–
–
–
–
–
D (106 cells/ml)
T (106 cells/ml)
Samples
0.05
0.08
1.7
0.05–1.7
0.61 ± 0.94
–
–
–
–
–
1c
2b
3a
Range
Mean ± SD
T = tetraploid cells; D = diploid cells; HNC = haploid noncondensed types; HNC1 = fluorescence intensity (FI) 13–14; HNC2 = FI 11–12.9; HNC3 = FI 8–10.9;
HNC4 = FI 4.5–7.9, where the diploid fluorescence peak was 24.5; and HC = haploid condensed.
a
Non-obstructive azoospermia.
b
Extreme oligo-terato-asthenozoospermia
c
Oligo-terato-asthenozoospermia.
Table V. Quantitative estimation of cells in semen from azoospermic samples where flow cytometric histograms fell into pattern 5
H total (106 cells/ml)
0.08
1.05
2.6
11.9
12.8
98.7
0.08–8.7
21 ± 38
HC (106 cells/ml)
0.08
0.9
2.6
10
12
84
0.08–84
18.3 ± 29.7
HNC (106 cells/ml)
HNC4
HNC3
HNC2
HNC1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.15
–
1.9
0.8
–
0.15–1.9
0.95 ± 0.88
–
–
–
–
–
14.7
–
D (106 cells/ml)
T (106 cells/ml)
Samples
0.16
0.53
0.59
0.48
0.2
5.3
0.16–5.3
1.21 ± 2
0.02
0.05
–
0.14
–
1.1
0.02–1.1
0.32 ± 0.51
1b
2a
3b
4b
5b
6c
range
mean±s.d
T = tetraploid cells; D = diploid cells; HNC = haploid noncondensed types; HNC1 = fluorescence intensity (FI) 13–14; HNC2 = FI 11–12.9; HNC3 = FI 8–10.9;
HNC4 = FI 4.5–7.9, where the diploid fluorescence peak was 24.5; and HC = haploid condensed.
a
Non-obstructive azoospermia.
b
Oligo-terato-asthenozoospermia.
c
Severe hypomobility.
somatic origin (epididymal, deferential, prostatic cells, leukocytes, etc.). In accordance with the classification of patients
into normal fertile donor, OTA and azoospermia, significant
differences were found in the proportions of the types of cells
found in semen (Wald et al., 2004). Testicular sperm morphology and quality are reported to depend on the degree of their
maturation (Yavetz et al., 2001). These changes contribute to
the sperm chromatin condensation that decreases the penetrability of fluorescent markers such as PI into the DNA, resulting in decreasing intensities of red fluorescence as chromatin
condensation increases. Previous studies with testis single cell
suspensions (Golan et al., 2000; Janca et al., 1986) detected
four peaks on histograms: T (tetraploid), D (diploid), HNC
(non-condensed haploid) and MC (mature condensed haploid). Similar findings were observed with human semen cells
in this study.
Our results suggest the value of SFC in non-invasive investigation of cases of men with infertility problems. In seven out
of 17 cases in our validation study, more information was
obtained by SFC than by routine testicular histology. This may
be explained by the fact that SFC examines cells shed by all
parts of both testes, while histological examination surveys
only the small portions obtained by biopsy. In our opinion, in
mild or moderate cases of male infertility, routine semen
laboratory investigation yields enough information and SFC is
not required. It could be an additional diagnostic and prognostic
3474
tool in extreme OTA and in NOA before decision making
when an invasive testicular sperm retrieval procedure may
be required. The retrieval success rate of these surgical procedures is only ∼40% (Kahraman et al., 1996; Mulhall et al.,
1997; Westlander et al., 1999; Friedler et al., 2002) and
remains poorly predictable. The husband’s age, FSH or
inhibin-B levels and testicular volume are poor predictors
(Tournaye et al.,1997; Gil-Salom et al., 1998; Friedler et al.,
2002; Soffer, 2004). Even genetic disorders such as Klinefelter
syndrome or Y long arm AZFc (Silber and Repping, 2002)
gene deletions are not able to predict the presence or absence
of spermatozoa. Deletions in regions AZFa and AZFb predict testicular sperm extraction (TESE) failure (Brandell
et al., 1998; Silber and Repping, 2002), but these are very
rare. We badly need a simple, non-invasive and reliable
assay to help clinicians counsel patients with severe male
infertility before referring them for testicular surgery to
locate spermatozoa for ICSI. SFC appears to be a very good
candidate for such an assay.
Acknowledgements
The authors acknowledge the skilled technical assistance of Sarita
Kaufman and Anna Umansky. The work was supported, in part, by a
grant to R.G. from the office of the Chief Scientist, Ministry of
Health, State of Israel and the Minz Lau fund, Tel Aviv University.
Flow cytometry of human semen
References
Brandell RA, Mielnik A, Liotta D, Ye Z, Veeck LL, Palermo GD and Schlegel PN
(1998) AZFb deletions predict the absence of spermatozoa with testicular
sperm extraction: preliminary report of a prognostic genetic test. Hum
Reprod 13,2812–2815.
Coskun S, Tbakhi A, Jaroudi KA, Uzumcu M, Merdad TA and Al-Hussein KA
(2002) Flow cytometric ploidy analysis of testicular biopsies from spermnegative wet preparations. Hum Reprod 17,977–983.
Dey P, Mondal AK, Singh SK and Vohra H (2000) Quantitation of spermatogenesis by DNA flow cytometry from fine-needle aspiration cytology
material. Diagn Cytopathol 23,386–387.
Evenson DP, Jost LK, Baer RK, Turner TW and Schrader SM (1991) Individuality of DNA denaturation patterns in human sperm chromatin structure
assay. Reprod Toxicol 5,115–125.
Fossa SD, Melvik JE, Juul NO, Pettersen EO and Theodorsen L (1989) DNA
flow cytometry in sperm cells from unilaterally orchiectomized patients
with testicular cancer before further treatment. Cytometry 10,345–350.
Friedler S, Raziel A, Strassburger D, Schachter M, Soffer Y and Ron-El R (2002)
Factors influencing the outcome of ICSI in patients with obstructive and nonobstructive azoospermia: a comparative study. Hum Reprod 17,114–121.
Gillan L, Evans G and Maxwell WMC (2005) Flow cytometric evaluation of
sperm parameters in relation to fertility potential. Theriogenology 63,445–457.
Gil-Salom M, Romero J, Minguez Y, Molero, MD, Remohi J and Pellicer A
(1998) Testicular sperm extraction and intracytoplasmic sperm injection: a
chance of fertility in non-obstructive azoospermia. J Urol 160,2063–2067.
Giwercman A, Clausen OP, Bruun E, Frimodt-Moller C and Skakkebaek NE
(1994) The value of quantitative DNA flow cytometry of testicular fineneedle aspirates in assessment of spermatogenesis: a study of 137 previous
maldescended human testes. Int J Androl 17,35–42.
Golan R, Weissenberg R, Oschry Y, Shochat L and Lewin LM (2000)
Spermatogenesis in the golden hamster during the first spermatogenic
wave: a flow cytometric analysis. Mol Reprod Dev 55,205–211.
Graham JK (2001) Assessment of sperm quality: a flow cytometry approach.
Anim Reprod Sci 68,239–247
Hacker-Klom UB, Gohde W, Nieschlag E and Behre HM (1999) DNA flow
cytometry of human semen. Hum Reprod 14,2506–2512.
Janca FC, Jost LK and Evenson DP (1986) Mouse testicular and sperm cell
development characterized from birth to adulthood by dual parameter flow
cytometry. Biol Reprod 34,613–623.
Kahraman S, Ozgur S, Alatas C, Aksoy S, Balaban B, Evrenkaya T, Nuhoglu A,
Tasdemir M, Biberoglu K, Schoysman R, Vanderzwalmen P and Nijs M
(1996) High implantation and pregnancy rates with testicular sperm extraction and intracylasmic sperm injection in obstructive and non-obstructive
azoospermia. Hum Reprod 11,673–676
Katoh C, Kitajima S, SagaY, Kanno J, Hurii I and Inoue T (2002) Assessment of
quantitative dual-parameter flow cytometric analysis evaluation of testicular
toxicity using cyclophosphamide- and ethinylestradiol-treated rats. J Toxicol Sci 27,87–96.
Kostakopoulos A, Protoyerou V, Tekerlekis P, Georgoulakis J, Louras G and
Goulandris N (2002) DNA flow cytometric, histological and hormonal analysis of Sertoli cell only syndrome (SECOS). Int Urol Nephrol 33,77–79.
Mulhall JP, Burgess CM, Cunningham D, Carson R, Harris D and Oates RD
(1997) Presence of mature sperm in testicular parenchyma of men with nonobstructive azoospermia: prevalence and predictive factors. Urology 49,91–96
Ron-El R, Strassburger D, Friedler S, Komarovski D, Bern O, Soffer Y and
Raziel A (1997) Extended sperm preparation: an alternative to testicular sperm
extraction in non-obstructive azoospermia. Hum Reprod. 12,1222–1226.
Silber SJ and Repping S (2002) Transmission of male infertility to future generations: lessons from the Y chromosome. Hum Reprod Update 8,217–229.
Soffer Y (2004) Nonobstructive azoospermia; predictive factors of testicular
sperm retrieval and risks of assisted fertilization [Azoospermies non obstructives; facteurs prédictifs du prélèvement testiculaire et risques de la fécondation assistée] (French, English abstract). Andrologie (SALF) 14,34–44.
Suter L, Bobadilla E, Koch E and Bechter R (1997) Flow cytometric evaluation of the effects of doxorubicin on rat spermatogenesis. Reprod Toxicol
11,521–531.
Tournaye H, Verheyen G and Nagy PC (1997) Are there any predictive factors
for successful testicular sperm recovery in azoospermic patients? Hum
Reprod 12,80–86
Vigodner M, Lewin LM, Shochat L, Oschry I, Lotan G, Kleen B and Golan R
(2003) Evaluation of damage to the testicular cells of golden hamsters
caused by experimental cryptorchidism using flow cytometry and confocal
microscopy. Int J Androl 26,84–90.
Wald M, Lewin L, Soffer Y, Oschry Y, Shochat L, Wigodner M and Golan R.
(2004) Seminal cell characterization using flow cytometry in infertile men.
HaRefuah 143,22–25.
Weissenberg R, Golan R, Shochat L and Lewin LM (2002) Procarbazine
effects on spermatogenesis in golden hamster: a flow cytometric evaluation.
Arch Androl 48,91–100.
Westlander G, Hamberger L and Hanson C (1999) Diagnostic epididymal and
testicular sperm recovery and genetic aspects in azoospermic men. Fertil
Steril 14,118–122.
World Health Organization (1999) Laboratory Manual for the Examination of
Human Semen and Sperm–Cervical Mucus Interaction, 4th edn. Cambridge
University Press.
Yavetz H, Yogev L, Kleiman S, Botchan A, Hauser R, Lessing JB, Paz G and
Gamzu R (2001) Morphology of testicular spermatozoa obtained by testicular sperm extraction in obstructive and nonobstructive azoospermic men and
its relation to fertilization success in the in vitro fertilization-intracytoplasmic sperm injection system. J Androl 22,376–381.
Submitted on January 19, 2005; resubmitted on July 11, 2005; accepted on
July 13, 2005
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