1 Clinic and Policlinic of RadiotherapyRadiooncology, 2 Institute of Radiobiology, 3 Institute of Reproductive Medicine and 4 Department of Obstetrics and Gynaecology, University of Münster, Germany
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Abstract |
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Key words: DNA flow cytometry/light microscopy/male infertility/semen
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Introduction |
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Since the possibility of a long-term decline in human sperm concentration is highly controversial (Lerchl and Nieschlag, 1996), prospective studies on large population groups will be necessary to test the hypothesis. These could be facilitated by application of an automated method for sperm counting such as flow cytometry (FCM). Since this allows the analysis of thousands of spermatozoa, statistical errors are minimized.
Among the numerous studies on sperm populations in mammals (e.g. Harrison, 1997), only some of those reporting FCM analysis of semen samples relevant to the present study need be mentioned here. Measurement problems resulting from the asymmetric shape and the highly condensed chromatin (CC) of the spermatozoa were finally overcome: Zante introduced papain for the decondensation of sperm chromatin and used a flow cytometer less susceptible to optical-geometric artefacts (Zante et al., 1977), while Dean improved the measuring accuracy by hydrodynamic orientation of spermatozoa (Dean et al., 1978
). Further studies followed (Otto et al., 1979a
; Johnson et al., 1993
; Ashwood-Smith, 1994
; Fugger et al., 1995).
The reduced fluorescence intensity of stained sperm DNA when using epi-illumination systems allows reliable differentiation between haploid spermatozoa and haploid round spermatids and between diploid spermatozoa and other diploid cells (early germ cells or somatic cells such as leukocytes or epithelial cells) (Van Dilla et al., 1977; Otto et al., 1979b
; Hartmann et al., 1982
). Cell sorting of testicular cells and subsequent DNA FCM using experimental animals has been used to confirm the identity of the cells in each DNA histogram peak (Hacker-Klom et al., 1989
). Since the histograms obtained from both testicular and ejaculated cells are qualitatively similar in various different mammals including the human, the conclusions drawn from the sorting data may also be applicable to man. Clinical application of FCM as a suitable method for supplementing the information obtained from the spermiogram of patients with infertility was proposed by Otto et al. (1979b).
In the present study, we demonstrate that FCM provides information about the condensation state of chromatin and the ploidy status of spermatozoa additional to the data obtained by conventional light microscopy. Sperm counts are obtained by FCM. A sample of 155 patients with infertility was analysed and classified into eight groups with differing sperm DNA histograms. To the best of our knowledge this is the first 10 year follow-up study revealing subfertile patient groups by DNA content of spermatozoa.
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Materials and methods |
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All female partners were seen for infertility work-up in the Department of Obstetrics and Gynaecology of the University. No ovulatory disorders were revealed in the 155 women. In two women bilateral occlusion was detected by laparoscopy. In-vitro fertilization was therefore performed.
FCM
Aliquots of the 171 ejaculates were fixed with 10% acetic acid for 24 h to 2 months at 5°C. The semen samples were prepared slightly modified from the previous protocol (Otto et al., 1979a). Thus 0.2 ml of fixed cells were diluted in 3 ml DAPI solution (5 µg 4',6-diamidino-2-phenylindole 2·HCl/ml 0.1 mol/l Tris-HCl buffer, pH 8.0, with 40 mmol/l MgCl2). In special cases, the fixed cells were centrifuged for 10 min at 200 g, resuspended in 1 ml 0.1 N sodium citrate-sodium hydroxide buffer, pH 6.4, containing 300 Anson units papain/ml (Anson unit: 1 unit will produce a
a280 of 0.001 per min at pH 2.0 at 37°C, measured as TCA-soluble products using haemoglobin as substrate), 20 mM dithioerythritol (DTE), 1 mmol/l ethylenediaminetetraacetic acid (EDTA) disodium salt and 1% dimethylsulphoxide (DMSO) and incubated for 15 min at 20°C. The samples were centrifuged again, resuspended and stained with DAPI as above.
A PAS II flow cytometer (Partec GmbH, Münster, Germany) equipped with a mercury arc lamp (HBO 100; Osram, Munich, Germany), a UG1 excitation filter, KG1 and BG38 heat filters, a TK420 beam splitter, and a GG435 barrier filter (Schott Glas, Mainz, Germany) in front of the photomultiplier was used. Each sample was measured at least twice.
Analysis of frequency histograms
Approximately 2x104 cells were measured for each frequency histogram. DNA frequency histograms were evaluated using cumulative frequency distributions (Figure 1). We discriminated one category more than Fosså's group (Fosså et al., 1989
), i.e. five categories: (i) cells with sub-haploid DNA content <1CC (debris that may be of apoptotic origin, Darzynkiewicz et al., 1992). The term `CC' means `condensed chromatin' and is used to indicate haploid spermatozoa which have a normal DNA content of IC but which is too condensed to be stained accordingly (Zante et al., 1977
); (ii) mature haploid spermatozoa in the 1CC peak; (iii) haploid round spermatids in the 1C peak; (iv) diploid spermatozoa in the 2CC peak; and (v) cells registered to the right of the 2CC level including 2C cells (leukocytes, G1-spermatogonia, primary spermatocytes at preleptotene etc.), cells in the DNA synthesis phase (S) and 4C cells (primary spermatocytes etc.; cf. Zante et al., 1977, Fosså et al., 1989).
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Spermiograms
Ejaculates were analysed by light microscopy according to WHO (1987).
Statistics
Correlation coefficients between different features of spermatozoa as determined by FCM and light microscopy were determined for identical samples with the method of least square deviations, using a programmable calculator assuming a linear regression. Since neither the correlation coefficient nor regression analysis was considered appropriate in the analysis of measurement method comparison, a graphical technique was applied (Bland and Altman, 1986). The
2 test with Yates' correction for continuity for contingency tables and the unpaired t-test were applied wherever appropriate. In general, data are given as mean ± SD.
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Results |
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Class 3 contained 10 histograms with <70% mature haploid spermatozoa at 1CC. The typical DNA histogram (Figure 4d) shows a peak representing haploid mature spermatozoa comprising 67% of the particles counted, a high amount of debris (31%), a few haploid round spermatids, diploid spermatozoa and no cells with a DNA content higher than 2CC. The ejaculate is oligozoospermic (3.3x106 spermatozoa/ml) and teratozoospermic with only 17% normal spermatozoa. A semen analysis of the same patient ~4 months earlier yielded a similar spermiogram. FCM analysis assigned this earlier sample to class 5.
Histograms of classes 48 characterized different types of spermatogeneic perturbations. Class 4 has diploid mature spermatozoa at 2CC at a high level of at least 5% and >70% 1CC. The typical histogram showed 6.3% diploid spermatozoa (Figure 4e). This ejaculate was normozoospermic with 72x106 spermatozoa/ml. In all, 47 samples fell into this class (Table I
). Within class 4, there were 8.6 ± 3.5% diploid spermatozoa compared with only 2.5 ± 0.72% within group 1, and 2.1 ± 0.54% double-headed spermatozoa were identified by LM in group 4 in contrast with only 0.6 ± 0.35 within group 1. The difference is significant at the P < 0.05 level by t-test.
Class 5 was characterized by a reproducible skewing of the 1CC peak to the left (Figure 4f). Although the skewing could not be changed by treating the samples with papain for 1560 min (thus inducing sperm chromatin decondensation), no explanation other than a disturbance of chromatin condensation of the haploid spermatozoa that could make the spermatozoa resistant to papain could be found. In this ejaculate (Figure 4f
) 55% of the haploid spermatozoa were not properly condensed. The patient had a parvisaemia (pathologically small ejaculate volume <2 ml), with an ejaculate volume of only 1.2 ml, was normozoospermic with 40x106/ml, and had 39% normal spermatozoa. Sixteen histograms fell into this class (Table I
). Only 45% of the samples within group 5 were normozoospermic.
Typical of class 6 is that >10% cells were >1CC, indicating the presence of immature germ cells and/or inflammatory cells. The representative histogram of class 6 (Figure 4g) shows 68% haploid spermatozoa, 8% debris and other cell types: spermatogenic cells, including haploid round spermatids (9%), diploid spermatozoa (5%), and cells with a regular 2C DNA content (4%), e.g. leukocytes and macrophages indicating infection. The spermiogram revealed that the ejaculate is oligoteratozoospermic (7.6x106 spermatozoa/ml with only 12% normal spermatozoa). In one extreme case, the histogram resembles a typical DNA frequency distribution of mammalian testicular tissue (including that of man) with four peaks (Figure 4h
). In the ejaculate, there were only 0.4x106 spermatozoa/ml and 23% round cells as revealed by LM. Sixteen samples fell into this category (Table I
). Within this group, there were only 29% normozoospermic samples and, on average, more amorphous spermatozoa than normal forms. The frequency of round cells was highest in this group: 9.4 ± 1.0x106/ml in contrast with only 3.9 ± 0.8x106/ml in group 1.
Class 7 contained no or only a few spermatozoa/ml. No complete histogram could be obtained even if the whole sample was measured (Figure 4i). As revealed by light microscopy, the sample came from a cryptozoospermic teratozoospermic ejaculate (0.7x106 spermatozoa/ml, total volume 5 ml containing 2x106/ml round forms, and 37% normal spermatozoa in the sediment). Sixteen samples fell into this category. The spermiograms revealed a mean of only 2.2x106 spermatozoa/ml among samples within this group (Table I
).
Class 8 was characterized by a skewing of the particle distribution from channel 0 onwards, indicating a preponderance of cellular debris in the representative DNA histogram (Figure 4j). This ejaculate, with a volume of 7.0 ml, pH 7.5, was totally azoospermic. Ten samples fell into this category (Table I
). None of these samples was normozoospermic. The spermiograms revealed that any spermatozoa present were amorphous rather than normal, and some defect tails were found.
Table I summarizes the results of the 171 ejaculates assigned to classes 18: only 7% of the patients with fertility problems are in the class with the highest sperm quality (class 1). Overall, the patients presented with a mean of 68 ± 23% haploid spermatozoa, a mean sperm density of 33 ± 44x106/ml and a mean of 41 ± 18% normal spermatozoa. In all, 13% of the patients naturally fathered a child during the 10 years of observation. Only 4% of the men with
5% diploid spermatozoa (class 4), 17% of class 6 and none of the class 5 patients with malcondensed spermatozoa or class 7 and 8 patients with low sperm concentrations or high amounts of cellular debris in the semen respectively fathered children. The two cycles of in-vitro fertilization resulted in a pregnancy in one woman whose husband belonged to class 3. The husband of the woman not achieving a pregnancy by in-vitro fertilization belonged to class 4. The
2 test reveals a statistically significant difference in paternity rates between the classes (P = 0.02). When classes 13 and 48 are compared, the difference is highly significant (P < 0.001).
Assuming a linear correlation, the percentage of 1CC cells in the DNA histogram was correlated best with the percentage of normal spermatozoa in the spermiogram (r = 0.67) (Table II). Debris in the histograms were correlated most favourably with spermatozoa with defective tails (r = 0.74), diploid spermatozoa with double-headed spermatozoa (r = 0.96), 2C cells with leukocytes (r = 0.97), and skewing of peak I with amorphous sperm heads (r = 0.76).
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Discussion |
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As has been shown in the present paper, FCM allows precise determination of sperm concentration. Linear associations between the sperm concentration obtained by conventional LM and by FCM for normozoospermic men and for oligozoospermic patients with counts down to 0.1x106 spermatozoa/ml respectively indicate that counting by FCM may be performed reliably, rapidly and objectively.
The 155 patients enrolled in our study had a mean sperm concentration of 32.6 ± 43.7x106/ml. The average sperm concentration ±SD of 68 ± 23% within the group of 155 patients as analysed by FCM was lower than that indicated by Fosså et al. (1989) for patients with testicular cancer (91%) and the corresponding control group (97%), reflecting decreased semen quality in patients with impaired fertility.
However, sperm counts are not the only parameter determining fertility (cf. Bostofte et al., 1982). Since the evaluation of motility and morphology by LM tends to be subjective (WHO, 1987), other parameters of fertility need to be found.
In the present paper, eight classes with different semen quality were determined by FCM and correlated with the results of the spermiograms. About one-third of the 155 patients were normozoospermic, with other factors having apparently affected the fertility. Increasing fractions of up to 35% of cellular debris of possibly apoptotic origin did not affect fertility in normo- and oligozoospermic men (classes 13). One unexpected feature, however, was that only two of the 47 men with >5% diploid spermatozoa fathered children within 10 years (class 4). Although these patients had high mean sperm concentrations and high percentages of normal haploid spermatozoa, the occurrence of a high frequency of diploid spermatozoa probably reflects not only faulty meiotic segregation involving the synaptonemal complex, but also major disturbances of spermatogenesis (Weissenberg et al., 1998). In certain patients, an abnormally high frequency of diploid spermatozoa seems to be correlated with repeated miscarriages (de Geyter et al., 1997
), possibly caused by sperm polyploidy. Polyploidy, mainly triploidy, is found in 20% of human spontaneous abortions (Tolksdorf, 1979
). In cases with repeated abortions, sorting of diploid spermatozoa may therefore be useful.
It was also striking that there were no paternities among the 18 class 5 patients with malcondensed sperm during the 10 year follow-up: malcondensation of sperm chromatin is considered to be correlated with infertility not necessarily reflected by poor sperm morphology (Evenson and Melamed, 1983; Evenson et al., 1991
; Hofmann and Hilscher, 1991
; Golan et al., 1997
; Spanò et al, 1998
). The detection of disturbances in murine sperm chromatin condensation after very low-dose X-rays (Sailer et al., 1995
) suggests that screening for genetic damage could be useful in the diagnosis and characterization of infertility. Another reason for the skewing of the sperm histogram, besides malcondensation of sperm chromatin, might be apoptosis (as suggested by Dr Z.Darzynkiewicz, personal communication).
Within class 6, the presence of immature germ cells or inflammatory cells did not affect fertility within the 10 year observation period. It was no surprise that the 16 patients with very low sperm concentration of zero or close to zero (class 7) and the 10 men with cellular fragments prevailing in the ejaculates (class 8) were infertile.
As suggested by these data, DNA FCM might be helpful in future studies of human infertility, allowing the clinician to assess rapidly and objectively the sperm count, the state of chromatin condensation and the presence of immature germ cells, of inflammatory cells and of diploid spermatozoa or of aneuploid tumour cells in the ejaculate (cf. Otto et al., 1979b; Clausen and Åabyholm, 1980; Evenson et al., 1980; Åabyholm and Clausen, 1981; Steen and Hanson, 1981; Evenson and Melamed, 1983; Fosså et al., 1989; Evenson et al., 1991; Seligmann et al., 1994; Golan et al., 1997; Weissenberg et al., 1998). Changes induced intentionally (e.g. male contraception) or accidentally during treatments (e.g. therapies employing radiation, hormones or cytostatic agents and affecting spermatogenesis) can be readily monitored and documented. Environmental or occupational toxin exposure might be suggested by identifying suspected decreases in the quality of semen in epidemiological analyses (cf. Spanò et al., 1983, 1998).
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Acknowledgments |
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Notes |
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References |
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Submitted on January 13, 1999; accepted on June 9, 1999.