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From the Departments of * Obstetrics and
Gynecology, Division Reproductive Medicine, and
Urology, University Medical Centre Nijmegen,
the Netherlands.
| Correspondence to: Ms L. Ramos, Department of Obstetrics and Gynecology, Division Reproductive Medicine, (intern post 415), University Medical Center Nijmegen. PO Box 9101, 6500 HB Nijmegen, the Netherlands (e-mail: l.ramos{at}obgyn.umcn.nl). |
| Received for publication September 24, 2003; accepted for publication December 18, 2003. |
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Abstract |
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Key words: Sperm selection, sperm normality, chromatin, condensation, DNA stainability
One approach to obtaining information about the adequacy of the ICSI criteria in selecting "normal sperm" from (non)ejaculated samples is to assess the sperm normality and DNA integrity of the sperm fraction selected for injection. To obtain further insight into the quality of spermatozoa, we adapted a computerized karyometric image analysis (CKIA) system to evaluate the sperm nucleus (Ramos et al, 2002a). This method is based on the stainability of the cells with a DNA-specific stain (Feulgen) in combination with a computer image analyzer system. The advantage of the CKIA system is the integral and objective assessment of the head features of human spermatozoa. CKIA analyzes a series of parameters that are related to different aspects of the nucleus: morphometry (eg, form, size), DNA stainability, and chromatin texture characteristics.
In this study, we focused on the evaluation of epididymal sperm from obstructive azoospermic (OA) patients. In these samples, there was a mixture of sperm with different maturational stages. In addition to immature and mature sperm, with incomplete and complete DNA condensation, respectively (Saowaros and Panyim, 1979; Evenson et al, 1986), postmature and damaged cells may be present in epididymal samples. Whether the maturational differences can be recognized using the ICSI selection criteria (morphology and motility) is unknown.
In a previous report concerning the evaluation of sperm markers and DNA integrity, we found that motility is a good marker for the selection of sperm with low damage evaluated with the terminal deoxynucleotidyl transferase–mediated deoxyuridine-5'-triphosphate nick end labeling (TUNEL) assay (Ramos and Wetzels, 2001). This finding was also valid for motile epididymal sperm from OA patients (Ramos et al, 2002b). The goal of the present study was 1) to examine the nuclear characteristics of epididymal spermatozoa from OA patients; and 2) to evaluate whether the selection criteria routinely used with ICSI (normal morphology and motility) are adequate markers for the selection of "normal" spermatozoa in ejaculated and epididymal samples according to the CKIA criteria.
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Materials and Methods |
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All patients signed an informed consent for participation in the study. The project was approved by the Ethical Committee of the University Medical Centre Nijmegen (UMCN, CMO) and the Dutch Central Committee for Research Involving Human Subjects (CCMO, The Hague).
Sperm Samples
Epididymal and donor sperm samples were processed as described previously
(Ramos et al, 2002b). Sperm
samples were washed in human tubal fluid medium (Cambrex; Bio Whittaker
Europe, Vervier, Belgium) supplemented with 10% human plasma proteins (CLB,
Amsterdam, the Netherlands) and were concentrated by centrifugation at 500
x g for 5 minutes.
From patients and donors, a droplet of the washed sperm samples (total/unselected population) was allowed to air dry on a glass slide. The rest of the sperm samples were used for the selection of spermatozoa using the ICSI criteria. For this purpose, we mimicked the routine ICSI procedure, except that selected sperm were placed on a glass slide for further evaluation. Briefly, 1 µL of sperm suspension was placed in 5 µL of a 5% polyvinylpyrrolidone solution (Medicult, Jyllige, Denmark) and left to swim out for 2 to 5 minutes. Using a magnification of 400x, morphologic normal motile spermatozoa were aspirated using micromanipulators connected to an ICSI injection pipette (Humagen, Charlottesville, Va). This procedure was repeated until enough sperm (50–100) were collected for analysis. The selected sperm fraction was allowed to dry before the fixation and staining procedure was continued.
Feulgen Stain
All chemicals were provided by Merck, Darmstadt, Germany, unless indicated
differently. Air-dried sperm samples were prefixed in a freshly prepared
Carbowax/NaCl 0.9% solution (1:1 [vol/vol]) (100% Carbowax: 2%
Polyethyleneglycol MW 1500 in 50% ethanol) for at least 24 hours (at 4°C).
Subsequently, the samples were fixed for 1 additional day with 100% Carbowax
(at 4°C) and then immersed in Böhm solution (10% formalin, 5% acetic
acid glacial, and 85% methanol) for 2 hours. After fixation, nuclei were
stained according to the Feulgen-Schiff reaction (hydrolysis in 5 N HCl for 60
minutes and stain in Schiff reagent for 30 minutes at room temperature). The
slides were mounted in Permount (Fischer Scientific, Fairlawn, NJ).
The CKIA Method
The measurement specifications of the CKIA method have been extensively
described previously (van der Poel et al,
1992; Ramos et al,
2002a). Cytomorphologic measurements were made using a microscope
connected to a CCD-video camera (Vision Technology, Eindhoven, the
Netherlands). The system consists of a frame grabber board (VFG frame grabber;
Image Technology, Woburn, Mass) connected to a personal computer. Using
1000x magnification, images of 512 x 512 pixels were captured,
digitized, and stored in the computer before analysis. The images were
corrected for background and shading, and they were filtered before applying
local segmentation. Each cell image was then processed independently from the
images of other cells. The nuclear boundary was delineated and separated from
the background. The nuclei were automatically analyzed and numbered, enabling
"postanalysis" verification of the objects. After computer
analysis, each detected nucleus was visually screened, and artifacts or faulty
segmented nuclei were eliminated. The karyometric parameters recorded for each
cell were grouped into the following 3 categories:
The codes and description of all karyometric parameters measured are summarized in Table 1. The normal ranges are based on the mean ± 1 standard deviation obtained from previous assessments from normospermic donors (Ramos et al, 2002a). Cryopreservation of the sperm samples did not influence the outcome of CKIA (data not shown), but special attention is required to achieve stabilization of the light source during the CKIA assessment.
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In this study, we defined the qualitative analysis of a sperm as the value assessed for each parameter per cell (evaluation of each parameter in a spermatozoon). A cell was considered "normal" when the values assessed for all parameters analyzed were within the predefined normal ranges. The quantitative analysis was defined as the frequency of "normal" sperm in each category (morphometry, DNA stain, and chromatin texture) and in the summed categories. This result is expressed as the percentage of normal sperm per sample.
Statistical Analysis
Differences between the samples were analyzed by the 1-way analysis of
variance and the Mann-Whitney U test when corresponding.
P-values <.05 were considered statistically significant. Data were
analyzed using the SPSS 11.1 software package (SPSS Inc, Chicago, Ill).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Quantitative Analysis of the CKIA
The biologic interpretation of the assessed parameters is complex and
difficult to apply in the clinical evaluation of male infertility. Therefore,
sperm "normality" per category or sample was also calculated. The
quantitative CKIA analysis involves the combination of normal values for all
individual parameters, and it is expressed as the percentage of normal
spermatozoa per category (or per sample). In
Table 3 and the Figure, the
results of the quantitative analysis (grouped into the categories of
morphometry, DNA stainability, and chromatin texture and the combination of
these categories) are presented. The Figure helps to visualize these results
and also shows the median and extreme values. No statistical differences were
found in the unselected (original) samples between patients and donors in any
category (Figure). The ICSI selection of epididymal and ejaculated sperm
resulted in an increase in the frequency of normal spermatozoa in each
category (Table 3), yielding
comparable morphologic features in both patients and donors. However, the
improvement in the frequency of "normal" sperm after selection was
significantly higher in donor samples than in patient samples in chromatin
texture and DNA stainability and, consequently, in the total sample
(Figure).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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One possible explanation for the relatively low numbers of sperm scoring "normal" with CKIA relies on the normal ranges used. The normal ranges described in our previous study (Ramos et al, 2002a) were derived from the mean ± 1 standard deviation obtained from sperm samples that were previously classified as normospermic by World Health Organization criteria; however, the CKIA assessment is based on a principle that is different from that of the standard morphology. Moreover, normospermia is not necessarily equivalent to fertility. Validation and, eventually, adjustment of these normal ranges need to be performed in a much larger fertile population. On the other hand, in the perspective of the present study, emphasis is placed on the outcome of nuclear differences of the same samples previous to or after selection and not the accuracy of the normal values.
A remarkable finding of the qualitative measurements is that only the
parameters related to the morphometry were significantly different between
donors and patients in unselected sperm samples. After selection, however, the
inverse was found: epididymal and ejaculated sperm samples differed in all
parameters, except for the area and the IOD (morphometric-related parameters).
This finding suggests that morphology selection at a magnification of
400x is adequate for the selection of similar-sized sperm but that it
does not discriminate between other nuclear features. We conclude that
spermatozoa with similar external characteristics do not necessarily hold the
same internal structures. This points to a shortage in the routine sperm
selection and needs further
investigation.
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Contrary to our expectation, a lower OD and IOD mean value was found for epididymal sperm. For human sperm, the OD of the Feulgen stain is related to the accessibility of the stain in the nuclear chromatin (Bito et al, 1999), which is higher in immature sperm (Saowaros and Panyim, 1979). Thus, an increase in the OD of the sample should be found. This was not always the case in caput spermatozoa from OA patients. A larger variation in the staining within samples and between OA patients was found, indicating a large heterogeneity in chromatin packaging in these samples. We speculate that in OA men, a fraction of the epididymal samples are "hypercondensed" sperm (aged). These postmature or aged spermatozoa may have continued thiol crosslinking, presenting some degree of chromatin hypercondensation. Hypercondensed nuclei are less accessible for Feulgen stain, leading to our present results.
Taking into account the results of the quantitative analysis for each CKIA category (see Table 3; Figure), patient and donor samples did not differ before selection. After selection, a larger frequency of "normal" spermatozoa was found in both patients and donors, but the proportion of normal sperm in the donor samples was significantly higher than in the latter group. ICSI selection increases the frequency of "normal" sperm as assessed by the CKIA system, but the improvement differs per sperm sample and origin. We should also take into account that the freeze-thaw procedure may act as an extra selection for the donor sperm samples, increasing the frequency of sperm with normal chromatin compaction.
In conclusion, an increase in the frequency of morphologic normal spermatozoa as evaluated by the CKIA method was observed after selection by morphology and motility under ICSI conditions. This finding joins the results of our previous work, where motile-selected spermatozoa showed an increase in the nuclear integrity assessed by TUNEL (Ramos et al, 2002b). The present results give rise to further research on sperm selection procedures for ICSI, effective chromatin packaging, and DNA integrity in spermatozoa from different origins.
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Acknowledgments |
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Footnotes |
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References |
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Bartoov B, Berkovitz A, Eltes F. Selection of spermatozoa with
normal nuclei to improve the pregnancy rate with intracytoplasmic sperm
injection. N Engl J Med. 2001; 345:1067–1068.
Bito M, Czihak G, Betz S, Wastian E, Frick J. Differences in the DNA-stainability of spermatozoa from fertile and suspected infertile men. Int J Androl. 1999; 22:275–281.[Medline]
Evenson D, Darzynkiewicz Z, Jost L, Janca F, Ballachey B. Changes in accessibility of DNA to various fluorochromes during spermatogenesis. Cytometry. 1986; 7:45–53.[Medline]
Morris ID, Ilott S, Dixon L, Brison DR. The spectrum of DNA damage
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Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992; 340:17–18.[Medline]
Ramos L, Hendriks JC, Peelen P, Braat DDM, Wetzels AMM. Use of
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Ramos L, Kleingeld P, Meuleman E, van Kooy R, Kremer J, Braat D, Wetzels A. Assessment of DNA fragmentation of spermatozoa that were surgically retrieved from men with obstructive azoospermia. Fertil Steril. 2002b; 77:233–237.[Medline]
Ramos L, Wetzels AMM. Low rates of DNA fragmentation in selected
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Saowaros W, Panyim S. The formation of disulfide bonds in human protamines during sperm maturation. Experientia. 1979; 35:191–192.[Medline]
Tesarik J, Mendoza C, Greco E. Paternal effects acting during the
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van der Poel HG, Schaafsma HE, Vooijs GP, Debruyne FM, Schalken JA. Quantitative light microscopy in urological oncology. J Urol. 1992; 148:1–13.[Medline]
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