1 Oregon Regional Primate Research Center, and 2 Departments of Cell-Developmental Biology and Obstetrics-Gynecology, Oregon Health Sciences University, 505 N.W. 185th Avenue, Beaverton, OR 97006, USA and 3 Department of Obstetrics and Gynecology, Tohoku University School of Medicine, 11, Seiryou-machi, Sendai, Miyagi, 980-8574, Japan
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Abstract |
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Key words: male factor infertility/SUTI/ubiquitin
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Introduction |
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Materials and methods |
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Flow cytometry
Sperm suspensions were fixed, blocked and incubated with antibody KM-691, washed by resuspension in PBS and centrifugation, then incubated with FITC-conjugated goat anti-mouse IgM (dilution 1/80; Zymed), washed and resuspended in 500 µl of pure PBS without serum. Blank, negative control samples were prepared for each experimental sample by omitting the primary antibody. Typically, samples from five patients and one fertile donor were prepared and analysed in each session. Data from three representative sessions are shown. Samples were analysed using FACS Calibur Analyzer (Becton Dickinson, San Diego, CA, USA) at 488 nm. Relative levels of fluorescence in individual cells were recorded. A sample of PBS buffer used for labelling was used to eliminate non-specific fluorescence and to calibrate the cytometer; a blank sample of corresponding patient/donor was run before each anti-ubiquitin-labelled sample. Five thousand cells (5000 gated events) were recorded for each sample. Analysis threshold was set at channel zero. The median value, representing the channel number about which half the cells are dimmer and half the cells are brighter and increases with the increase in the number of brighter cells, was recorded. Other parameters taken into consideration were: (i) shape of the fluorescence histogram, typically with a sharp peak in fertile men and with flat shape in subfertile men; (ii) scattering pattern reflecting cell shape, typically with a single focus in fertile men with uniform shape and with two foci or elongated large single focus in subfertile men with misshapen spermatozoa; (iii) shift of the curve towards high relative fluorescence in subfertile men; and (iv) position of the peak of relative fluorescence curve on the x-axis, typically close to 101 in fertile men and close to 102 in patients with elevated ubiquitin levels. Flow cytometry data in subfertile men typically displayed the combination of two or more of the above characteristics as described for patients, in addition to a high median value. After each repeat, leftover samples were stored overnight at 4°C and re-run the next day. No significant differences were found between such re-runs and original, first run data.
Electron microscopy
Sperm were isolated, fixed in formaldehyde and processed with antibody KM-693 without permeabilization as described for immunofluorescence, except that the fluorescently conjugated secondary antibodies were replaced by a 10 nm-gold-conjugated goat anti-mouse IgM (Jackson Immunochemicals) and the sperm were handled by centrifugation instead of being attached to microscopy coverslips. The labelled cells were pelleted by centrifugation, fixed for transmission electron microscopy (TEM) and embedded in Epon 812 as described previously (Sutovsky et al., 1999b). Ultrathin sections were cut on a Sorvall MT-5000 ultramicrotome (Ivan Sorvall Inc., Norwalk, CT, USA), stained by uranyl acetate and lead citrate, and examined in Philips EM 300 electron microscope. Negatives were scanned by Umax Powerlook 3000 flat bed scanner (Umax Technologies Inc., Fremont, CA, USA) and printed on a Sony UP-D 8800 dye sublimation printer using Adobe Photoshop 4.0 software.
Statistical analysis
Flow cytometry medians are shown for each of the three, non-overlapping groups in which the samples were processed for flow cytometry. Scatter diagrams and predicted linear regression lines were drawn based on sample regression coefficients (byx <;
>), calculated using flow cytometry medians and clinical data (sperm count, sperm motility, fertilization rate, cleavage rate) provided by the clinic (Snedecor and Cochran, 1973
; Sokal and Rohlf, 1981
). Correlation between flow cytometry medians and clinical parameters was evaluated by calculating sample correlation coefficients (r = <1;1>) as described (Snedecor and Cochran, 1973
). Median values for standard samples were not included in these calculations since the clinical data were not made available by the distributor of those samples.
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Results |
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By immunofluorescence, most spermatozoa from fertile donors displayed typical ellipsoid shape of the sperm head and a straight sperm tail with a mitochondrial sheath of even diameter. Weak ubiquitin labelling was found on the surface of the sperm tail's principal and end pieces in such sperm (Figure 1A), and, in some cases, on the equatorial segment of the sperm head (Figure 1B
). No permeabilization was used to avoid the contribution of constitutively ubiquitinated sperm substrates such as mitochondrial membrane proteins (Sutovsky et al., 1999a
, 2000a
) and nuclear histones (Chen et al., 1998
; Baarends et al., 1999
) to the fluorescent signal. Abnormal spermatozoa with strongly ubiquitinated, coiled or lasso tails were also found in the spermatozoa from both fertile donors (Figures 1C, 2A
), while their abundance, as judged subjectively by fluorescence microscopy was diminished in comparison with patient's samples. Abnormalities in the defective sperm (Figures 1, 2
) included coiled or lasso tails (Figure 1D
), misshaped and globozoospermic sperm heads (Figure 1E
), twin tails and/or twin heads (Figure 1
F,G), and spermatogenic and somatic cells (Figure 1H
), residual bodies (Figure 1I
) and fragmented residual cytoplasm (Figure 1J
) present in the ejaculates. Semen from donor S1 was used as a standard sample for flow cytometry, where its median channel for fluorescence peak (further `median'), reflecting relative number of sperm with average relative fluorescence, fluctuated between 17.15 to 22.88 (Figure 2A
). The presence of ubiquitin-cross-reactive proteins on the surface of defective spermatozoa was confirmed by ultrastructural immunocytochemistry (Figure 3AF
).
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Malformations of the sperm heads were the major anomaly in sperm of patient 2, while the sample contained mostly normal sperm. Accordingly, the infertility was previously diagnosed as tubal, with no pregnancy but excellent fertilization and cleavage rates (both 83.3%) and sperm characteristics (136.3 x 106/ml; 88.3%). Flow cytometry was not performed.
Patient 3 displayed significantly higher median (40.68 versus 22.88 in S1) and a shift in number of highly fluorescent cells (Figure 2B) by flow cytometry. By immunofluorescence, the major defect revealed was swollen sperm heads and lasso tails. Heads separated from tails, nuclear vacuoles and cytoplasmic droplets were also frequent. Infertility was deemed idiopathic with good sperm motility (88.3%) and indication for primary sterility. No pregnancy was obtained. SUTI indicates the diagnosis of male factor infertility in this previously unexplained case.
In patient 4, the major defect was large amounts of cellular debris in the form of irregularly shaped clusters (see Figure 1J), while most spermatozoa did not display major defects. Flow cytometry median channel was 27.38 as opposed to 22.88 in S1, and a significant shift in fluorescence distribution was observed. Both tubal and male factor infertility were previously diagnosed, with slightly reduced sperm count (34 x 106/ml) and low motility (23.7%). Male factor infertility was confirmed by SUTI.
The major defect in patient 5 was the presence of residual cytoplasmic bodies, normally removed by Sertoli cells in the testis, in the ejaculate (see Figure 1I). Nuclear vacuoles and swollen sperm heads were also frequent. Flow cytometry median of fluorescence peak was elevated to 28.39 versus 18.43 in S1. Sterility was previously diagnosed as tubal, with average sperm count of 61.7 x 106/ml and motility of 59.9%. No pregnancy was obtained despite the treatment for tubal infertility. SUTI suggests the contribution of previously undiagnosed male factor.
The prevailing anomaly in patient 6 was the presence of small cells with nuclei, probably leukocytes and/or immature spermatogenic cells (Figures 1H, 2C). Swollen sperm heads were also frequent. Median channel of fluorescence peak was 29.43 versus 22.88 in S1 with a shift towards highly fluorescent cells. Sperm count was high (256 x 106/ml), while only 25% of sperm were motile. Ubiquitin data confirm male factor infertility, diagnosed previously.
Patient 7 had a combination of swollen sperm heads, abnormal mitochondrial sheaths and residual cytoplasmic bodies. Other defects included nuclear vacuoles, cytoplasmic droplets still attached to the sperm mid piece, abnormal mitochondrial sheaths and large somatic cells present in the sample. Median reached 33.98 versus 18.43 in S1, with histogram shifted towards highly fluorescent cells. Previously diagnosed as a primary, tubal infertility, sperm count was good (222 x 106/ml), whereas motility was average (42.3%). SUTI suggests male factor contribution to this case previously diagnosed as maternal infertility.
Patient 8 carried a high number of misshapen sperm heads, suggesting a defect of the sperm nuclear condensation or differentiation of the perinuclear theca. Some abnormal mitochondrial sheaths and somatic cells were also observed. Median was high (39.24 versus 22.88 in S1) with a curve shift and a bifocal scatter pattern. This was an unexplained infertility with high sperm count (173 x 106/ml) and motility (67.6%), good fertilization rate (66.7%), but a low cleavage rate (27.3%). No pregnancy was obtained and SUTI indicated male factor.
Ubiquitinated somatic cells, probably leukocytes, were detected in the sample from patient 9 (Figure 2D). Other abnormalities included round and elongated spermatids and globozoospermic sperm present in the ejaculate. High median (35.23 versus 18.43 in S1), flat and shifted flow cytometry histogram, and multi-focal scattering patterns were recorded. Infertility was previously diagnosed as tubal, with average sperm count (70 x 106/l) and motility (54.2%). SUTI suggests male factor contribution.
Sample 10 contained ubiquitinated sperm with coiled tails, nuclear vacuoles and abundant residual cytoplasmic bodies. Both male factor and tubal infertility were diagnosed previously and are corroborated by subjective ubiquitin assay (flow cytometry was not performed). While the sperm count was good (145 x 106/ml), motility was only 25.2%. The remaining motile spermatozoa yielded identical cleavage and fertilization rates (66.7%).
Patient 11 had a good sperm sample with few ubiquitinated spermatozoa. No predominant abnormality was detected, although the abnormalities described in other cases were occasionally found. While the flow cytometry histogram was atypically flat and the sample displayed enlarged scatter pattern, the median was only 12.41 versus 17.15 in S1, and there was no shift. This case was previously diagnosed as tubal infertility, fertilization and cleavage rates were excellent (both 100%). Accordingly, SUTI did not indicate male factor.
The sample from patient 12 contained relatively few ubiquitinated sperm cells. The major defect observed was nuclear vacuoles. Median (21.29) was close to that of S1 (18.43) and the histogram was neither shifted nor flat (Figure 2E). Sperm parameters were excellent (189 x 106/ml; 87.3% motility). Considering the excellent results of SUTI and good cleavage rates (50%), male factor is not likely to contribute to this case of unexplained infertility.
The prevalent defect in patient 13 was the presence of twin heads and/or twin tails. Median channel of fluorescence peak was only 20.54, as compared to 18.43 in S1, while the histogram was flat and clearly shifted to the right, with a bifocal scattering pattern. The presence of ubiquitinated twin sperm may account for relatively low motility (47%) and 0% fertilization and cleavage rates. The case was pronounced idiopathic with no pregnancy. With the exception of a good median, ubiquitin data suggest male factor.
Patient 14 had a good sample with a major defect being broken and coiled tails (Figure 2F). This man had the lowest median value in sampled group (11.97 versus 17.15 in S1). The couple was diagnosed with tubal infertility, fertilization and cleavage rates were good (57.1 and 42.9% respectively). Motility was average (45.6%) with a good sperm count of 174 x 106/ml. There was no pregnancy and SUTI suggests that this infertility was not contributed by male factor.
Patient 15 carried a mixture of various defects, including lasso tails, swollen heads, nuclear vacuoles, cells, residual cytoplasm and abnormal mitochondrial sheaths. Median reached 33.98 versus 22.88 in S1, with a shift towards highly fluorescent cells. This case was previously diagnosed as idiopathic with good sperm parameters (140 x 106/ml; 78% motility), while SUTI points to a male factor infertility.
Abnormal, lasso and twin sperm tails were the prevailing defects in patient 16, other defects included malformed sperm heads and cells present in ejaculate. Both tubal and male factor infertility were diagnosed previously and corroborated by immunofluorescence data and low motility (33% at 213 x 106 sperm/ml), and 0% fertilization and cleavage rates. No pregnancy was obtained.
The major defect in patient 17 was nuclear vacuoles, while round sperm heads and a residual cytoplasmic droplet were also observed. The case was previously pronounced unexplained and a low cleavage rate (25%) was contradicted by a good IVF rate (83.3%) and sperm parameters (184 x 106/ml; 73.6% motility). Ubiquitin data suggest male factor contribution, while maternal contribution cannot be ruled out.
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Discussion |
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The present data show that the levels of sperm-surface ubiquitination corroborate available clinical profiles in 17 patients studied, in six of them confirming the previously diagnosed male factor infertility (patients 1, 4, 6, 10, 16), in five cases revealing possible reason for infertility that was previously assessed as idiopathic (patients 3, 8, 13, 15, 17), in three cases (patients 2, 11, 14) confirming the diagnosis of maternal only infertility and in three cases (patients 5, 7, 9) suggesting the contribution of previously overlooked male factor to the previously diagnosed maternal infertility. In patient 12, ubiquitin assays suggested that male factor is not likely to contribute to this case of idiopathic infertility. As expected, ubiquitin data were not correlated with treatment outcome, as pregnancy was not obtained in some couples with good sperm-ubiquitin parameters (2, 11, 12, 14; most of them tubal infertility cases) and some couples with increased sperm ubiquitination did achieve pregnancy after IVF (4, 9, 10).
Both immunofluorescence and flow cytometry assays provided valuable information about each given sperm sample. The main value of the subjective immunofluorescence screening is in its ability to reveal which particular types of sperm defects prevail in a given sample. This could be useful in planning the treatment strategy. For example, immunofluorescence assay revealed sperm head malformations reminiscent of globozoospermia, a clinical indication for intracytoplasmic sperm injection (ICSI) combined with artificial oocyte activation (Rybouchkin et al., 1997), in patient 1. Both fertilization and cleavage rates were zero and pregnancy was not obtained. Similar defects were overlooked in patient 17 during clinical semen evaluation and the infertility was originally diagnosed as idiopathic. Other easy-to-overlook causes of presumed idiopathic infertility, such as the easily decapacitated spermatozoa defect (Kamal et al., 1999
), Kartagener's syndrome (Baccetti et al., 1980
) and dysplasia of fibrous sheath (Chemes et al., 1987
), can be recognized by epididymal ubiquitination machinery and properly diagnosed by SUTI (V.Rawe, P.Sutovsky and E.Neuber, unpublished data). Similarly, spermatozoa with head defects in azh/azh mutant mice (Cole et al., 1988
; Meistrich et al., 1990
), display strong surface-ubiquitination (P.Sutovsky and R.Moreno, unpublished data). Subjective immunofluorescence examination readily detects the elevated number of ubiquitinated spermatozoa, somatic and spermatogenic cells, and residual cytoplasm, and after appropriate training, the evaluators could also generate quantitative data by counting the number of highly ubiquitinated spermatozoa per unit of spermatozoa screened (e.g. per 1000 spermatozoa in 10 randomly selected optical fields).
Ideally, the subjective immunofluorescence assay should be complemented by flow cytometry trial providing the means for objective, unbiased quantification of ubiquitin titre in a sperm sample. Such analysis could be performed using conventional flow cytometers or dedicated, automated semen analysers (Coetzee et al., 1999a,b
). Other immunological titration assays, such as enzyme linked immunoabsorbent assay (ELISA) could be used to objectively evaluate the ubiquitination levels of human sperm samples and eventually developed into simple titration test kits detecting increased ubiquitin levels in fresh semen samples. All fluorescence based assays could be streamlined by using anti-ubiquitin antibodies conjugated with suitable fluorochromes. Alternative flow cytometric assays of sperm morphology were proposed, including the sorting of semen samples stained with DNA stains (Evenson and Melamed, 1983
; Filatov et al., 1999
; Hacker-Klom et al., 1999
), or specific antibodies against single-stranded DNA (van der Schans et al., 2000
), apoptotic markers (Sakkas et al., 1999
) and leukocyte contaminants (Moilanen et al., 1999
). Other objective assays of sperm quality are based on the detection of sperm DNA damage resulting from apoptosis, oxidation or necrosis (Baccetti et al., 1996
; Shen et al., 1999
; Irvine et al., 2000
). Most abnormal sperm cells revealed by such assays will likely be surface ubiquitinated and thus also detectable by SUTI, while our method will also detect abnormalities of the sperm tail and cellular contaminants other than defective sperm, including leukocytes, residual bodies, cellular detritus, persisting cytoplasmic droplets, and immature spermatogenic cells present in patients' ejaculates. Finally, sperm cell fragments generated during sample processing (e.g. tails and heads separated by pipetting and centrifugation) will not be detected as defective by SUTI, unless they originate from abnormal sperm.
In patients 3, 13, 15 and 17, relatively good sperm count, motility, fertilization and cleavage parameters did not suggest male factor infertility, while ubiquitin data suggested male factor contribution to these cases of unexplained infertility. This is probably due to the fact that defective and immotile spermatozoa were removed from such ejaculates prior to IVF (but not before clinical semen analysis and SUTI) by gradient centrifugation or swim-up, thus yielding good fertilization rates. The surface-ubiquitinated spermatozoa may interfere with motility and/or fertilizing ability of such spermatozoa after coitus, but can be eliminated by motile sperm separation prior to IVF. It follows that the fertilization rate in vitro may not be a reliable parameter for the diagnosis of male factor in such unexplained cases. The predictive and diagnostic value of other traditional sperm parameters, such as sperm concentration, motility and morphology has been disputed (Tomlinson et al., 1999). Accordingly, the analysis of regression and correlation in the subgroup of patients tested by flow cytometry (Figure 4
) showed low-to-moderate positive correlation of sperm ubiquitination to sperm count, sperm motility and fertilization rates (motility > count > fertilization; sample correlation coefficients r = 0.337, 0.268 and 0.046 respectively), while there was a substantial negative correlation (r = 0.432) between ubiquitin, flow cytometry median and cleavage rates. Three patients, in whose samples SUTI ruled out the contribution of male factor (11, 12, 14), had low flow cytometry medians, and average-to-excellent sperm count, sperm motility, fertilization rates and cleavage rates. As a method reflecting the occurrence of morphological abnormalities in individual sperm samples, SUTI may have predictive value for the outcome of IVF treatment, as measured by embryo cleavage rates. Other studies showed that sperm morphology has higher predictive value than other clinical sperm parameters, when strict criteria are applied (Lim et al., 1998
; Zinaman et al., 2000
). Donnelly et al. (1998) reported correlation coefficients between strict morphology and IVF outcome, that are similar to our data on the correlation between ubiquitin medians and cleavage rates. Therefore, ubiquitin data may be closely correlated with abnormal morphology, thus providing a new tool for automated semen analysis. Further tests on larger groups of samples from subfertile patients with known clinical history and previous strict morphology assessments will be performed in order to determine the exact correlation between these parameters. It is not likely that SUTI analysis will be highly predictive of pregnancy rates after IVF, since these are affected by maternal factors independent of sperm quality (e.g. tubal infertility, implantation rate). The tendency towards the increased sperm count and motility in patients with increased sperm ubiquitination could be due to the saturation of the epididymal mechanism responsible for the removal of defective, ubiquitinated spermatozoa.
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In summary, the objective immunoassay of sperm-surface ubiquitination provides a promising new approach to semen evaluation in humans. In contrast to the existing, subjective methods of semen evaluation discussed by others (Amann, 1989; Coetzee et al., 1999a
,b
), SUTI assay may provide a reliable, objective and measurable parameter of abnormal spermatogenesis and suboptimal semen quality acquired prior to sperm discharge from urogenital tract. Of particular importance is the ability of the SUTI assay to detect male factor in some cases of infertility, previously pronounced idiopathic.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 30, 2000; accepted on October 19, 2000.