1 Fertility-Assisted Medical Center, Laboratoire Unilab, Clinique Monplaisir, 810 avenue Frères Lumière, 69008 Lyon, 2 Assistance Medical Center Grenoble-Belledonne, 83 avenue Gabriel Peri, 38400 St Martin d'Heres, France and 3 University of Missouri-Columbia, S141 ASRC, 920 East Campus Drive, Columbia, MO 65211-5300, USA
4 To whom correspondence should be addressed. Email: sutovskyp{at}missouri.edu
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Abstract |
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Key words: andrology/infertility/male factor/sperm/ubiquitin
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Introduction |
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Male factor represents 40% of all infertility cases worldwide, with additional male infertility cases possibly being misdiagnosed as idiopathic infertility (Kim and Lipshutz, 1999
; Schultz and Williams, 2002). While conventional semen evaluation by light microscopy provides a good estimate of sperm quality and fertility in most cases, more accurate, biochemical tests are sought in reproductive medicine to better evaluate the sperm quality and to properly diagnose male infertility (reviewed by Eliasson, 2003
). Ubiquitin is a suitable marker of male infertility owing to its association with the surface of defective epididymal and ejaculated spermatozoa (reviewed by Sutovsky, 2003
). While it is also possible to measure ubiquitin in the human seminal plasma (Lippert et al., 1993
), only the sperm-surface ubiquitin correlates with fertility (Sutovsky et al., 2001b
, 2004
). The sperm ubiquitin tag immunoassay (SUTI) has been developed primarily as a flow cytometric assay that is highly sensitive, but requires expensive equipment and a trained, experienced operator. Here, we adapted the immunofluorescence-based SUTI assay for simple microscopic screening of human semen samples in a clinical setting.
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Materials and methods |
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For all analyses, the gradient-separated spermatozoa were thawed in warm water (37°C) and washed by centrifugation through Universal IVF medium (Medicult, Limonest, France). Available clinical data included original clinical diagnosis of infertility (male factor, idiopathic, female), treatment (IVF or ICSI), number of oocytes retrieved, number of oocytes with the first polar body, number of two-pronuclear zygotes (2PN) with the second polar body (PB+) at 1618 h after IVF/ICSI, number of cleaved embryos at 4446 h after insemination, number of transferred and cryopreserved embryos at 72 h and pregnancies. Available semen quality parameters, obtained prior to gradient separation included sperm count, sperm motility, sperm morphology (David's classification; David, 1975; Jouannet et al., 1988
), percentage of acrosome-reacted spermatozoa after induced acrosome reaction and percentage of ubiquitin-immunoreactive spermatozoa.
The sperm parameters of the male patients were analysed by conventional light microscopic semen evaluation using WHO criteria for sperm count and motility, and WHO morphology criteria adapted according to David (1975). By David's classification, a semen sample was considered fertile at a minimum of 30% normal spermatozoa of Test-Simplet staining (Boehringer, Mannheim). Patients were divided into four groups as follows.
Group 1 (n=28): male factor infertility. The sperm parameters showed one or more anomalies of the spermogram, as defined by WHO/David's criteria. These patients were included in an ICSI protocol, except for two patients whose spouses underwent intrauterine insemination. Female partners of all 28 men had normal clinical fertility profile.
Group 2 (n=27): idiopathic, unexplained infertility. The clinical parameters of the men and women in this group did not reveal any reproductive problems, and clinical sperm parameters appeared normal by WHO/David's criteria. None of the patients had children previously.
Group 3 (n=21): female infertility with unknown male fertility. Female patients from these couples were diagnosed with one or more types of reproductive dysfunction, including endometriosis, tubal factor, anovulatory infertility, polycystic ovary syndrome and uterine malformations. Male clinical parameters were normal, but no previous pregnancies were reported for the partners of these men.
Group 4 (n=17): female infertility with male patients with history of proven fertility. This group was similar to group 3, but all male partners had children in their previous relationships. Nevertheless, it is possible that sperm quality in some of these subjects declined since the time they fathered their children.
Evaluation of acrosome reaction, acrosomal integrity and sperm viability
Spermatozoa were separated on discontinuous density gradient as described above, washed in IVF medium, adjusted to final concentration of 520 x 106/ml and incubated for 4 h at 37°C under 5% CO2. To induce acrosome reaction, 100 ml of sperm in IVF medium were incubated with 100 ml of calcium ionophore A23187 solution (2 mmol/l final concentration; SigmaAldrich, Saint-Quentin Fallavier, France) for 30 min at 37°C under 5% CO2. To assess sperm viability, spermatozoa were washed in phosphate-buffered saline (PBS), centrifuged and the pellet was mixed with 200 ml of PBS containing 1 mg/ml Hoechst 33258 (DNA stain; Hoechst/Aventis, Strasbourg, France) and incubated in the dark for 10 min. Spermatozoa were washed with PBS+2% PVP and resuspended in 100 ml of PBS. After washing, 500 ml of 1% formol solution was added to each tube, incubated for 5 min at room temperature, centrifuged, resuspended in 100 ml of PBS, smeared on a microscopy slide, air-dried and fixed with acetone for 30 min at 4°C. Acrosomal integrity was determined by incubation with FITC-conjugated peanut agglutinin (PNA)-lectin (lectin from Arachis hypogaea conjugated with FITC, 1 mg/ml in deionized water; Sigma) as described by Cross et al. (1986), Kallajoki et al. (1986)
and Mortimer et al. (1987)
. Briefly, a 2 ml stock solution of PNA lectin was mixed with 50 ml of ultrapure water, and 20 ml of this solution was added on a slide with fixed spermatozoa and incubated for 15 min at 37°C in a humidified chamber. Slides were rinsed in PBS, air-dried and covered with a coverslip in a drop of mounting medium. Sample evaluation was performed under an epifluorescence microscope with a 100x lens using 340 nm wavelength filter for Hoechst 33258 (blue fluoresce of dead spermatozoa) and a 490 nm filter for PNA-FITC (green fluorescence of intact sperm acrosomes). In each sample, 200 live, Hoechst-negative spermatozoa were evaluated for percentage of acrosome reaction with and without induction.
Immunofluorescence SUTI assay
Two microlitres of gradient-purified sperm pellets from each subject were resuspended in a 500 µl drop of 37°C warm KMT medium on a poly-L-lysine coated microscopy coverslip and allowed to attach for 5 min on a slide warmer (Sutovsky, 2004). Coverslips were submerged in 2% formaldehyde in PBS and fixed for 40 min. Samples were blocked for 25 min in 5% normal goat serum (NGS; Sigma) in PBS and incubated for 40 min with the monoclonal antibody KM-691 raised against the recombinant human ubiquitin (dilution 1/100; Kamiya Biomedical Company, Seattle, WA, USA). PBS with 1% NGS was used for washing and dilution of primary and secondary antibodies. After washing, samples were incubated for 40 min with FITC-conjugated goat anti-mouse IgM (dilution 1/80; Sigma Diagnostics) and the DNA-stain DAPI (Sigma Diagnostics) was added to this solution 10 min before the end of incubation. Samples were washed and mounted on microscopy slides. Negative controls included omission of the anti-ubiquitin antibody and incubation with KM-691 immunosaturated with purified erythrocyte ubiquitin (Sigma). In both cases, microscopy and image acquisition setting comparable to parameters for ubiquitin image acquisition were used. Clinical sample analyses were performed by using a Leitz Laborlux 11 microscope with a fluorescent module (Ploemopak 2.5) using a 100x immersion lens. Additional analyses were carried out using a Nikon Eclipse 800 microscope with epifluorescence and differential interference contrast (DIC) optics, and a CoolSnap HQ CCD camera. All analyses (93 subjects) were carried out by the same evaluator, an experienced andrologist-MD. Using the available clinical grade equipment, it was difficult to categorize the levels of anti-ubiquitin fluorescence on the sperm tails. Therefore, the analysis was focused on the presence or absence of the ubiquitin signal on the sperm heads. Since the sperm samples were not permeabilized, it was presumed that the observed staining was due to anti-ubiquitin-immunoreactive proteins on the sperm head surface.
Flow cytometric SUTI assay
To rule out an effect of freeze-thawing on semen quality, sperm samples from two patients were washed by centrifugation through Universal IVF medium (Medicult) as described above (less the PureSperm gradient separation) and screened by flow cytometric SUTI as described previously (Sutovsky et al., 2001b, 2004
). Briefly, samples were fixed in 2% formaldehyde in PBS, blocked with 5% NGS and incubated sequentially in suspension with anti-ubiquitin KM-691 and goat-anti-mouse IgM-FITC. Ubiquitin levels in 10 000 cells per sample were evaluated as described previously (Sutovsky et al., 2004
; see Figure 4).
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Results |
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Within the male factor group (Figure 3; Table I), in which all but two couples were treated by ICSI, percentage ubiquitylated spermatozoa correlated negatively with percentage cleaved/2PN embryos (r=0.42), percentage cleaved/PB+ (r=0.39) embryos and percentage cleaved/ovulated ova (r=0.37). Interestingly, there were strong negative correlations between the percentage of acrosome-reacted spermatozoa after induced acrosomal reaction and several embryo cleavage parameters within this group (r=0.36 to 0.78). Negative correlations between the percentage of ubiquitylated spermatozoa and individual embryo development characteristics were also found in group 3. Ubiquitin showed moderate levels of positive correlation with percentage dead/necrotic spermatozoa in all four groups combined (r=0.38).
To ascertain that increased rates of sperm ubiquitylation were not due to freeze-thawing of semen samples, we performed flow cytometric analyses (SUTI assay; Sutovsky et al., 2001b, 2004
) on semen samples from two donors before and after semen preparation and freeze-thawing (Figure 4). As expected, these analyses did not reveal a significant change in the pattern of sperm ubiquitylation before and after freeze-thawing (Figure 4). This is likely due to the fact that substrate ubiquitylation occurs through a stable covalent bond and specific deubiquitinating enzymes are required for the removal of polyubiquitin chains from the ubiquitylated substrates. In addition to flow cytometric analysis, we performed the simplified immunofluorescence SUTI as described above, and found that the percentage of spermatozoa with ubiquitylated sperm heads was 42 before and 34 after freeze-thawing in the first donor, and 38 before and 31 after freeze-thawing in the second donor (samples were not purified on PureSperm gradient).
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Discussion |
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The above observations were made despite the purification of spermatozoa on PureSperm gradient, which preceded the evaluation of ubiquitin content and presumably removed most of the defective, ubiquitylated spermatozoa found in raw semen samples. It follows that gradient separation alone may not be sufficient to completely deplete defective spermatozoa from a semen sample used for assisted reproduction. This has particular implications for ICSI, wherein one spermatozoon is chosen from such partially purified population based on morphological appearance and motility (if present). The pre-selection of spermatozoa for low ubiquitin could add a higher level of stringency to sperm selection for ICSI even after sperm separation on a density gradient. Such added level of stringency could be especially important in cases with very low sperm count and motility, wherein the gradient separation is not possible. Two such patients were initially present in group 1 in the present trial, but were eliminated from all statistical analyses since their sperm samples were not treated equally to the remaining 93 patients' samples. Both samples showed high percentages of ubiquitin-positive sperm heads (data not shown). The present data further support the requirement for a more stringent sperm selection by showing better IVF and ICSI fertilization rates in men with low sperm ubiquitin levels. It remains to be resolved how to avoid a potentially harmful fluorescence exposure of spermatozoa in such protocols. Since the ubiquitin binding occurs mainly on the surface of defective spermatozoa, a good alternative to fluorescent labelling of ubiquitylated spermatozoa could be the immunodepletion of gradient-separated sperm samples with matrix-bound anti-ubiquitin antibodies or the detection of defective spermatozoa by microspheres conjugated with anti-ubiquitin antibody or a ubiquitin-binding protein.
Higher levels of sperm ubiquitylation also corroborate poor sperm quality and fertility in the group of men diagnosed with male factor infertility (group 1), wherein sperm ubiquitin correlated negatively with the percentage of cleaved embryos, and percentage of embryos with two pronuclei after IVF or ICSI. As mentioned above, it was not possible to perform gradient separation in several additional patients originally included with this group, owing to low sperm concentration and poor motility. Therefore, it is possible that in such patients, some spermatozoa with a high proportion of surface ubiquitin could be selected during ICSI procedure, and this could adversely affect PN development and embryo cleavage rates after ICSI. This does not rule out a possibility of obtaining a pregnancy in such patients. Indeed, the average percentage of ubiquitylated spermatozoa was high for both pregnant and non-pregnant male factor couples, while the remaining three groups, predominantly treated by IVF, showed significantly lower ubiquitin averages in pregnant couples compared with non-pregnant ones. This could be reconciled by the use of ICSI in 26 of 28 male factor couples. High percentage of ubiquitylated spermatozoa could thus be an indication for the use of ICSI in idiopathic couples, whenever IVF is also considered. Therefore, it could be attempted to use the simplified ubiquitin-based semen evaluation to reduce the number of unsuccessful IVF cycles by redirecting the treatment towards ICSI.
In group 2 (idiopathic infertility), we found increased sperm ubiquitin levels after gradient separation in some of the subjects (eight out of 27 had >3% ubiquitylated sperm), but no significant correlation between sperm ubiquitin and embryo cleavage rates. This can be explained by two factors. First, at least half of these couples could have experienced infertility due to an undiagnosed female factor. Secondly, in couples with an undiagnosed male factor, just as in properly diagnosed male factor patients, the fertilization and development rates could have been improved by gradient separation of spermatozoa, which appears to be a fairly effective method for selecting the fittest cells for IVF in cases with acceptable sperm morphology and motility.
In groups 3 and 4, where the sperm parameters were expected to be normal by WHO/David's criteria, we found a significantly lower proportion of sperm ubiquitylation compared with groups 1 and 2. Similarly, there were no correlations between sperm ubiquitin and embryo development parameters. However, at least one patient in group 4 showed a high degree of sperm ubiquitylation.
Recent studies showed that a portion of spermatozoa in both fertile and infertile subjects contain a partially degraded nuclear DNA, probably a result of apoptosis or necrosis. Such DNA fragmentation can be detected by various techniques including SCSA, COMET or TUNEL. Late, but not early, paternal influence on embryo development is thought to be related to sperm DNA fragmentation (Benchaib et al., 2003; Henkel et al., 2003
; Larson-Cook et al., 2003
; Tesarik et al., 2004
). The DNA damage can be also detected by the flow cytometric sperm ubiquitin assays, in which a significant positive correlation between ubiquitin median values and median values of TUNEL-induced fluorescence is observed (Sutovsky et al., 2002
). In contrast to the above DNA/chromatin integrity assays, SUTI detects a variety of other defects, not only those related to DNA damage. Ubiquitin detection may thus be a reliable marker of overall sperm quality, including, but not limited to, DNA damage. The level of the sperm-surface ubiquitylation could be informative in the sperm evaluation using highly sensitive, objective flow cytometric or biochemical techniques, but also by using simple epifluorescence microscopy such as the method described here. This is useful even after sperm gradient separation/purification. Further studies of sperm ubiquitin and related infertility markers could lead to the development of new techniques for individual sperm selection for ICSI, which in turn could improve the rate of success in most couples with male factor infertility and in some couples with unexplained infertility.
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Acknowledgements |
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References |
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Submitted on June 11, 2004; resubmitted on March 1, 2005; accepted on March 8, 2005.
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