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 2 Male Infertility Unit, Assaf HaRofe Medical Center, Zerifin, Israel

3 Corresponding author. E-mail: rachelgo{at}post.tau.ac.il


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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)Go 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., 2000Go). Differences in flow cytometric histograms between control cell suspensions and those from cryptorchid testes (Vigodner et al., 2003Go) or from animals treated with procarbazine (Weissenberg et al., 2002Go) demonstrated inhibition at various loci of the spermatogenesis pathway. Similar studies done on testes of rats following cyclophosphamide and ethynylestradiol administration (Katoh et al., 2002Go) or doxorubacin (Suter et al., 1997Go) 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., 1994Go; Coskun et al., 2002Go). Kostakapoulos et al. (2002) and Dey et al. (2000)Go 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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, 1999Go) 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 x 106/ml, median 104 x 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-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., 1997Go); 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:

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.



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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.

 

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., 1997Go) 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 haematoxylin–eosin. 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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 ~108spermatozoa/ml comprising >99% of the cells present. Five patterns were seen in histograms of semen from infertile patients.



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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.

 

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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Table I. Quantitative estimation of cells in semen from azoospermic samples where flow cytometric histograms fell into pattern1

 

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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Table II. Quantitative estimation of cells in semen from azoospermic samples where flow cytometric histograms fell into pattern 2

 

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.


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Table III. Quantitative estimation of cells in semen from azoospermic samples where flow cytometric histograms fell into pattern 3

 

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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Table IV. Quantitative estimation of cells in semen from azoospermic samples where flow cytometric histograms fell into pattern 4

 

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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Table V. Quantitative estimation of cells in semen from azoospermic samples where flow cytometric histograms fell into pattern 5

 

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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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. (1999)Go 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 analysed. Our study adds this important information. Fossa et al. (1989)Go 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)Go. SFC was also used in the analysis of various defects of sperm for the assement of sperm quality and fertility potential (Graham, 2001Go; Gillan et al., 2005Go).

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)Go. 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., 2004Go). Testicular sperm morphology and quality are reported to depend on the degree of their maturation (Yavetz et al., 2001Go). 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., 2000Go; Janca et al., 1986Go) 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., 1996Go; Mulhall et al., 1997Go; Westlander et al., 1999Go; Friedler et al., 2002Go) and remains poorly predictable. The husband’s age, FSH or inhibin-B levels and testicular volume are poor predictors (Tournaye et al.,1997Go; Gil-Salom et al., 1998Go; Friedler et al., 2002Go; Soffer, 2004Go). Even genetic disorders such as Klinefelter syndrome or Y long arm AZFc (Silber and Repping, 2002Go) 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., 1998Go; Silber and Repping, 2002Go), 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on January 19, 2005; resubmitted on July 11, 2005; accepted on July 13, 2005.





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