1 Department of Clinical Biochemistry, Sackler Medical School, Tel Aviv University, Ramat Aviv and 2 Male Infertility Unit, Assaf HaRofe Medical Center, Zerifin, Israel
3 Corresponding author. E-mail: rachelgo{at}post.tau.ac.il
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Abstract |
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Key words: flow cytometry/male infertility/non-obstructive azoospermia/spermatogenesis
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Introduction |
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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 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.
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Materials and methods |
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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 1.56 ml, median 2.8 ml; sperm count range 36179 x 106/ml, median 104 x 106/ml; motility range 4060%, median 50%; motility grade (on a scale from 0 to 4) range 3.13.5, median 3.4; normal forms (strict criteria) range 611%, median 7%.
Of the 44 samples from infertile men, 10 were from oligo-terato-asthenozoospermic 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 x 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 non-obstuctive 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, 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 x 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:
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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 x400 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 material was routinely fixed in Stieve solution and sent to the Pathology Laboratory. Samples were embedded in paraffin, sectioned and stained with haematoxylineosin. Slides were observed under x400 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.
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Results |
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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 x 106cells/ml. A semen sample from a patient with obstructive azoospermia (subject 5) contained 0.33 x 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).
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Pattern 2 (Figure 2C)
Diploid and tetraploid cells without haploid cells. Quantification (Table II) showed a mean value of only 0.17 x 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.
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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 1314, HNC2 FI 1112.9, HNC3 FI 810.9 and HNC4 FI 4.57.9, 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.
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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.
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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.
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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.
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Discussion |
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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 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 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 husbands 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.
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Acknowledgements |
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References |
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Submitted on January 19, 2005; resubmitted on July 11, 2005; accepted on July 13, 2005.
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