Human Reproduction, Vol. 15, No. 8, 1717-1722,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques
1 Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, 2 Department of Obstetrics and Gynecology, University of Minnesota, Minneapolis, MN 55454 and 3 Department of Obstetrics and Gynecology, University of Nebraska Medical Center (UNMC), Omaha, NE, USA
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Abstract |
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The predictive value of sperm chromatin integrity for pregnancy outcome following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was studied in 24 men attending a university-based assisted reproductive techniques laboratory using the flow cytometric sperm chromatin structure assay (SCSA). The SCSA is a measure of the susceptibility of sperm DNA to low pH-induced denaturation in situ. The mean percentage of spermatozoa in the neat sample demonstrating DNA denaturation was significantly lower in the seven men that initiated a pregnancy (15.4 ± 4.6, P = 0.01) than in the 14 men who did not initiate a pregnancy (31.1 ± 3.2). No pregnancies resulted if
27% of the spermatozoa in the neat semen sample showed DNA denaturation. These data demonstrate that SCSA parameters are independent of conventional semen parameters. Furthermore, the SCSA may allow physicians to identify male patients for whom IVF and ICSI will be unlikely to result in pregnancy initiation.
Key words: chromatin structure/human fertility/in-vitro fertiliza- tion/intracytoplasmic sperm injection/SCSA
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Introduction |
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Identification of sperm parameters that predict the outcome of assisted reproductive techniques would allow physicians to counsel patients on their chance of pregnancy following IVF and intracytoplasmic sperm injection (ICSI). The value of this information has been the impetus for numerous studies designed to identify a single parameter or group of parameters that have significant predictive value for assisted reproductive techniques' (ART) outcomes. Sperm strict morphology criteria have been used effectively to develop thresholds that are predictive of fertilization and pregnancy rates following IVF (Kruger et al., 1986
; Vawda et al., 1996). In addition, better embryo quality has been associated with normal sperm morphology (Van der Zwalmen et al., 1991; Parinaud et al., 1993; Janny and Ménézo, 1994). Many investigators (Claassens et al., 1992; Sukcharoen et al., 1995; Hammadeh et al., 1997; Parinaud et al., 1997) have shown the importance of optimizing conventional sperm parameters, including morphology for IVF success. However, the importance of sperm morphology for ART's success has become increasingly ambiguous with the advent of ICSI which overrides many inherent boundaries for natural selection (Hoshi et al., 1996).
The ability of ICSI to bypass oocyte-associated boundaries has facilitated the successful treatment of males with severe oligozoospermia, teratozoospermia, azoospermia and immotile sperm cells (Engel et al., 1996). Some studies indicate that fertilization, embryo cleavage and pregnancy following ICSI are independent of conventional sperm parameters (Hammadeh et al., 1996; Oehninger, 1996). However, other studies have reported that impaired sperm quality, characteristic of ICSI patients, leads to a lower percentage of embryos that form blastocysts (Shoukir et al., 1998), poor blastocyst quality (Janny and Ménézo, 1994) and high abortion rates (Sanchez et al., 1996). Janny and Ménézo (1994) showed the strong paternal influence in preimplantation embryo development, reporting that both the overall number of blastocysts obtained and the number of patients having at least one blastocyst were reduced in patients with severely impaired sperm quality.
The influence(s) of sub-optimal sperm chromatin integrity on post-embryonic development is the subject of intense investigation. Spermatozoa from infertile men have a higher frequency of chromosomal abnormalities (Moosani et al., 1995), lower resistance to sodium dodecyl sulphate (SDS)-induced decondensation (Colleu et al., 1988), poor DNA packing quality (Hofmann and Hilscher, 1991; Bianchi et al., 1996; Filatov et al., 1999), increased DNA strand breaks (Lopes et al., 1998; Irvine et al., 2000) and susceptibility to acid-induced DNA denaturation in situ (Evenson et al., 1980; Evenson, 1999; Evenson et al., 1999; Spano et al., 2000). It is unclear if ART are effective in compensating for poor chromatin packaging and/or DNA damage or if sub-optimal chromatin integrity may contribute to the poor implantation rate (<20%) in the majority of ART patients (Edwards and Beard, 1999). Understanding of the effect of the paternal genome becomes more critical as we become less discriminatory with the maturity and quality of paternal nuclear material which is introduced into the oocyte (Sakkas, 1999).
The sperm chromatin structure assay (SCSA) holds promise for determining the importance of chromatin structure in ART outcomes. SCSA is an unbiased, quantitative assessment of sperm chromatin integrity defined as susceptibility of DNA to acid-induced denaturation in situ. SCSA parameters are stable within individuals over time if men are not exposed to reproductive stressors (Evenson et al., 1991). In addition, SCSA parameters are correlated with DNA strand breaks (Sailer et al., 1995; Aravindan et al., 1997) and decreased fertility in vivo (Evenson et al., 1980, 1999). However, SCSA parameters are not strongly correlated with World Health Organization (WHO) parameters including concentration, motility and morphology (Evenson et al., 1991). Therefore, SCSA parameters are independent and may have predictive value beyond WHO parameters for patient success in ART. The objective of this study was to examine the relationship between neat and washed semen sample SCSA parameters and fertilization, embryo grade and pregnancy outcome following ART.
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Materials and methods |
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Semen analysis
Semen samples were collected by masturbation from 24 men (mean age of 35 ± 4.8 years) after 2–7 days of abstinence and on the day of their partner's (mean age of 34 ± 5.1 years) oocyte aspiration for IVF or ICSI at the University of Nebraska Medical Center. Semen analysis was performed following WHO (1992) guidelines. Means for sperm concentration (x106/ml), motility (%), forward progression (%) and qualitative morphology (%) of neat and washed samples are shown in Table I
. Strict morphology of neat samples was also assessed for 17 of the patients during a semen analysis before the day of IVF or ICSI (Table I). No individuals with varicocele, obstructive and non-obstructive azoospermia, or underlying reproductive tract pathologies were included in the study population. Of the original sample of 24 men, six suffered from male factor infertility, two of whom achieved pregnancy.
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Sperm separation
A double-layered discontinuous Isolate© (Irvine Scientific, Santa Ana, CA, USA) gradient technique was used to prepare spermatozoa for IVF or ICSI. To form the gradient, 1 ml of the `lower layer' Isolate© (~90%) was pipetted into the bottom of a 15 ml conical centrifuge tube, and 1 ml of `upper layer' Isolate© (~45%) was pipetted gently over the bottom layer. Liquefied semen was placed on the top of the gradient using a sterile pipette, and the tube centrifuged at 200 g for 25 min. The supernatant was removed and discarded, and the sperm pellet suspended in 1 ml fresh HEPES-buffered human tubal fluid (HTF) containing 1% human serum albumin (h-HTF) (In Vitro Care Inc., San Diego, CA, USA). The suspension was centrifuged at 300 g for 3–5 min, and the supernatant aspirated and discarded. The sperm pellet was resuspended in h-HTF and a post-wash sperm analysis performed for concentration, motility and forward progression.
Sperm chromatin structure assay
Following the procedure(s) of Evenson and Jost (Evenson and Jost, 1994), the SCSA evaluated chromatin integrity of spermatozoa in neat and washed semen. 200 µl of sperm samples (1–2x106spermatozoa/ml) were treated for 30 s with 400 µl of a pH 1.2 solution containing 0.1% Triton X-100, 0.15 mol/l NaCl and 0.08 N HCl. Triton X-100 permeabilizes sperm cell membranes providing greater accessibility of acridine orange (AO) to DNA. The low pH solution partially denatures DNA in spermatozoa with abnormal chromatin structure. Spermatozoa with normal chromatin structure do not demonstrate DNA denaturation. After the 30 s acid treatment, 1.20 ml of AO staining buffer (6 µg AO/ml, 37 mmol/l citric acid, 126 mmol/l Na2HPO4, 1 mmol/l disodium EDTA, 0.15 mol/l NaCl, pH 6.0) was added to the cells before analysing by flow cytometry. AO that intercalates into double-stranded DNA (native; normal) fluoresces green (515–530 nm) while AO that associates with single-stranded (denatured) DNA fluoresces red (630 nm) when excited by a 488 nm light source (Darzynkiewicz et al., 1975).
The extent of sperm DNA denaturation was quantified using an Ortho Diagnostic Cytofluorograf II (Becton Dickinson, Westwood, MA, USA) with a closed quartz flow cell and a 100 mW argon ion laser operated at 35 mW power that was interfaced to a Cicero unit with PC-based Cyclops Software (Cytomation, Fort Collins, CO, USA). This system measured the amount of red and green fluorescence emitted from individual sperm cells flowing at ~200/s and calculated the 
t [red/(red+green) fluorescence] distribution and associated parameters for each sample. Sperm populations with normal chromatin structure have a small mean t (Xt), SDt, and percentage of cells outside the main population (COMPt, i.e. percentage of cells with denatured DNA). SCSA also identifies immature sperm nuclei by the percentage of cells with high green fluorescence (HGRN), reflecting uncondensed chromatin that is more accessible to the AO stain. Mature ejaculated spermatozoa have a 5-fold lesser DNA stainability than round spermatids (Evenson and Melamed, 1983).
Assisted reproductive techniques
Oocyte retrieval and incubation
Oocytes were retrieved from women, <40 years of age, undergoing ovarian stimulation [leuprolide acetate, FSH, and/or FSH/LH as detailed by Roy et al. (1998)] using ultrasound-guided transvaginal follicular aspiration. Oocytes were cultured in synthetic HTF-containing 1% human serum albumin (In Vitro Care Inc.) in a 5% CO2, 37°C, humidified incubator for ~16–18 h in the presence of washed spermatozoa (50 000 to 100 000 motile spermatozoa/ml) or after ICSI. ICSI was done using established techniques and only on mature oocytes (Palmero et al., 1992; Van Steirteghem et al., 1993).
Fertilization assessment and embryo culture
Oocytes were assessed for fertilization 16–18 h post-insemination or ICSI. For inseminated oocytes, cumulus cells were removed by repeated pipetting through a sterile, small bore, glass pipette. For inseminated and ICSI oocytes, fertilization was considered normal if two pronuclei and two polar bodies were identified. Oocytes without visible pronuclei were considered to be unfertilized. Oocytes with a single pronucleus or more than two pronuclei were considered to be abnormally fertilized and discarded. Patients were allocated to one or more of the following fertilization categories: normal (45% oocytes fertilizing normally), unfertilized (100% oocytes not fertilized), abnormal (20% oocytes fertilizing abnormally). Fertilized oocytes were transferred by pipette to a dish containing fresh culture medium (HTF plus 5% human serum albumin) and incubated for an additional 2 days.
Embryo grading
Assessment of embryo quality was made 2 days after fertilization using a modification of Veeck (1986) and the grading system was as follows. Grade 1: pre-embryo with blastomeres of equal size; no cytoplasmic fragments. Grade 2: pre-embryo with blastomeres of equal size; minor cytoplasmic fragments or blebs. Grade 3: pre-embryo with blastomeres of distinctly unequal size; few or no cytoplasmic fragments or pre-embryo with blastomeres of equal or unequal size; significant cytoplasmic fragmentation or pre-embryo with few blastomeres of any size; severe or complete fragmentation.
Embryo transfer
Tubal embryo transfer and uterine embryo transfer procedures were performed 3 days after fertilization. In general, tubal embryo transfer was used for women with normal Fallopian tubes (n = 11) and IVF and embryo transfer for women with tubal factors (n = 10).
Pregnancy outcome
Ultrasound detection for fetal sac was used to confirm a positive clinical pregnancy. One patient with only a positive quantitative ß-human chorionic gonadotrophin (ßHCG) was also included as a positive pregnancy.
Statistical analysis
2-Analysis was used to test the significance of SCSA parameter thresholds chosen for positive pregnancy. This analysis was applied to SCSA parameters of neat and washed samples separately. Each sample was analysed by SCSA twice, the replicate run immediately following the first. 2-Analysis was completed using the mean of the replicate SCSA values. Three patients who did not have embryos transferred due to abnormal fertilization and/or fertilization failure were not included in 2-analysis of pregnancy outcome. Four washed samples were removed from the statistical analysis because the amount and pattern of DNA degradation indicated delayed sample freezing. Two further washed samples were not included in SCSA analysis because of insufficient volume. If a relationship was identified through exploratory data analysis, regression analysis was used to determine the amount and significance of the correlation between SCSA parameters, WHO parameters and ART outcomes (Table II) by means of the SAS program (SAS, 1988).
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Results |
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Mean SCSA values are shown for neat and washed samples in Table III
. Spermatozoa in the neat semen of the seven men initiating pregnancy were significantly less susceptible to DNA denaturation than the 14 men not initiating pregnancy following embryo transfer (Table III). SCSA thresholds (Xt = 300, P < 0.01; SDt = 200, P = 0.01; and COMPt =27%, P < 0.01) for the susceptibility to low pH-induced denaturation in the neat sperm suspensions were predictive of no pregnancy (Table III). Pregnancy rate per oocyte transferred was 24% in patients with <27% (COMP t) of the spermatozoa in the neat sample showing acid-induced DNA denaturation compared to 0% pregnancy rate in patients with 27% denaturation (Table IV). The percentage of immature (HGRN) spermatozoa in the neat sample also appeared to have a threshold (17%) for pregnancy success; however, the confidence level was not significant (P = 0.19). No SCSA thresholds for the neat sample were identified for normal fertilization or embryo development (data not shown). However, four of the five patients with 20% abnormally fertilizing oocytes had SCSA parameters that exceeded the thresholds for successful pregnancy (Table IV). The mean percentage of spermatozoa showing dentauration (COMPt) in these five patients was 33% compared to 23% in the 19 patients with <20% abnormal fertilization.
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SCSA parameters of the washed sample were not predictive of fertilization, embryo development or pregnancy outcome (Figure 1
). No patient's washed sample exceeded the neat Xt or SDt thresholds, and only one patient's washed sample exceeded the neat COMPt threshold. In addition, no significant relationships between the SCSA parameters (neat or washed) and WHO parameters, fertilization or embryo development were identified by regression analysis.
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Discussion |
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The SCSA was predictive of negative pregnancy outcome when
27% (COMP t) of the spermatozoa in the neat sample showed acid-induced DNA denaturation. The percentage of cells with DNA damage was significantly (P = 0.01) greater in the 14 men with failed pregnancy outcome (31.1 ± 3.2) than in the seven men initiating pregnancy following IVF or ICSI (15.4 ± 4.6). Predictive thresholds for two other SCSA parameters [Xt (300), SDt (200)] were established for negative pregnancy outcome, indicating the potential value of SCSA analysis in an assisted reproduction treatment programme. Evenson et al. (1999) reported a similar threshold of 30% damaged spermatozoa (COMPt) for in-vivo fertility (Evenson et al., 1999), while Spano et al. (2000) reported that fecundity became small when >40% of the cells showed denaturation (Spano et al., 2000). Therefore, the SCSA parameters appear to provide an unbiased, quantitative assessment of sperm chromatin structure that is an independent predictor of in-vivo as well as in-vitro fertility potential, i.e. pregnancy. In contrast to flow cytometric SCSA results, Angelopoulis et al. (1998) reported that microscopic examination of AO fluorescence in unprocessed spermatozoa does not predict pregnancy outcomes following IVF cycles (Angelopoulis et al., 1998). The difference may be due to high intra- and interspecimen coefficients of variation reported for the microscopic AO method, making it less sensitive and less specific than the flow cytometric SCSA (Claassens et al., 1992).
There was no relationship between the susceptibility to DNA denaturation, assessed by the SCSA, and normal fertilization and early embryo development (Table IV
); however there was a trend for increased abnormal fertilization when DNA denaturation exceeded the established threshold. It was reported (Twigg et al., 1998) that DNA strand breaks did not affect the rate of fertilization. Similarly, spermatozoa with high amounts of DNA denaturation penetrated zona-free hamster eggs (Ibrahim and Pedersen, 1988), and spermatozoa with genetic-based defects fertilized normally (Engel et al., 1996). However, chromatin abnormalities appear to influence later embryonic development as demonstrated by the absence of clinical pregnancy in patients with SCSA parameters exceeding the thresholds established for neat samples.
SCSA parameters improved following density-gradient preparation as reported previously (Golan et al., 1997; Larson et al., 1999). However, the SCSA parameters of the prepared spermatozoa were not predictive of pregnancy outcome, indicating that elevated SCSA values in neat semen reflected chromatin abnormalities within the entire sperm population that were not eliminated by sperm preparation techniques. Other studies have shown that improved sperm concentration, motility and morphology after washing are not concomitant with increased ICSI fertilization and cleavage rates (Liu et al., 1994; De Vos et al., 1997). The current study demonstrated that ICSI leads to pregnancy in patients with poor sperm morphology and motility. For example, two couples with 
4% morphologically normal spermatozoa became pregnant following ICSI. Although the prognoses of these patients was poor based on conventional semen parameters, SCSA parameters of the neat semen were below the threshold for DNA damage (12 and 20% of spermatozoa showed DNA denaturation; i.e. COMPt), indicating that the chromatin integrity of the sperm population was adequate to support a viable pregnancy. These patients illustrate the importance of the additional, independent information provided by SCSA analysis. Other assessments of sperm chromatin structure including chromomycin A3 and acidic aniline blue staining are significantly associated with sperm morphology (Franken et al., 1999) and therefore may not provide the additional, independent information of the SCSA.
If applied clinically, the SCSA may assist the clinician in providing informed consent to patients, for example if donor spermatozoa should be considered. The SCSA will provide additional information, beyond conventional semen analysis, that may identify DNA damage that will not lead to viable pregnancies following transfer even though the spermatozoa may be capable of initiating fertilization and early embryo development.
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Acknowledgments |
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The authors thank Dr Paul Evenson for assistance with the statistical analysis. This work was supported in part by EPA Grant Number R820968-01, National Science Foundation Grants EPSCoR-9720642, OSR-9452894 and the South Dakota Future Fund. This is South Dakota Agricultural Experiment Station Publication Number 3127 of the journal series.
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Notes |
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4 To whom correspondence should be addressed at: Department of Chemistry and Biochemistry, Box 2170, South Dakota State University, Brookings, SD 57007, USA.E-mail: kjerstilea{at}hotmail.com
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References |
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Angelopoulos, T., Moshel, Y.A., Lu, L. et al. (1998) Simultaneous assessment of sperm chromatin condensation and morphology before and after separation procedures: effect on the clinical outcome after in vitro fertilization. Fertil. Steril., 69, 740–747.[Web of Science][Medline]
Aravindan, G.R., Bjordahl, J., Jost, L.K. et al. (1997) Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis. Exp. Cell Res., 236, 231–237.[Web of Science][Medline]
Bianchi, P.G., Manicardi, G.C., Urner, F. et al. (1996) Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa. Mol. Hum. Reprod., 2, 139–144.
Claassens, O.E., Menkveld R., Franken, D.R. et al. (1992) The acridine orange test: determining the relationship between sperm morphology and fertilization in vitro. Hum. Reprod., 7, 242–247.
Colleu, D., Lescoat, D., Boujard, D. et al. (1988) Human spermatozoal nuclear maturity in normozoospermia and asthenozoospermia. Arch. Androl., 21, 155–162.[Web of Science][Medline]
Darzynkiewicz, Z., Traganos, F., Sharpless, T. et al. (1975) Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp. Cell Res., 90, 411–428.[Web of Science][Medline]
De Vos, A., Nagy, Z.P., Van de Veld, H. et al. (1997) Percoll gradient centrifugation can be omitted in sperm preparation for intracytoplasmic sperm injection. Hum. Reprod., 12, 1980–1984.
Edwards, R.G. and Beard, H.K. (1999) Blastocyst stage transfer: pitfalls and benefits. Hum. Reprod., 14, 1–6.
Engel, W., Murphy, D. and Schmid, M. (1996) Are there genetic risks associated with microassisted reproduction? Hum. Reprod., 11, 2359–2370.
Evenson, D.P. (1999) Alterations and damage of sperm chromatin structure and early embryonic failure. In Jansen. R. and Mortimer, D. (eds), Towards Reproductive Certainty: Fertility and Genetics Beyond 1999. Parthenon Publishing Group, New York, pp. 313–329.
Evenson, D.P. and Melamed, M.R. (1983) Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry. J. Histochem. Cytochem., 31, 248–253.
Evenson, D.P. and Jost, L.K. (1994) Sperm chromatin structure assay: DNA denaturability. In Darzynkiewicz, Z., Robinson, J.P. and Crissman, H.A. (eds), Methods in Cell Biology, Vol. 42, Flow Cytometry. Academic Press, Orlando, FL, pp. 159–176.
Evenson, D.P., Darzynkiewicz, Z., and Melamed, M. (1980) Relation of mammalian sperm chromatin heterogeneity to fertility. Science, 210, 1131–1333.
Evenson, D.P., Jost, L.K., Baer, R.K. et al. (1991) Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod. Toxicol., 5, 115–125.[Web of Science][Medline]
Evenson, D.P., Jost, L.K., Zinaman, M.J. et al. (1999) Utility of the sperm chromatin structure assay (SCSA) as a diagnostic and prognostic tool in the human fertility clinic. Hum. Reprod., 14, 1039–1049.
Filatov, M.V., Semenova, E.V., Vorob'eva, O.A. et al. (1999) Relationship between abnormal sperm chromatin packing and IVF results. Mol. Hum. Reprod., 5, 825–830.
Franken, D.R., Franken, C.J., de la Guerre, H. et al. (1999) Normal sperm morphology and chromatin packaging: comparison between aniline blue and chromomycin A3 staining. Andrologia, 31, 361–366.[Web of Science][Medline]
Golan, R, Shochat, L., Weissenberg, R. et al. (1997) Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining. Mol. Hum. Reprod., 3, 47–54.
Hammadeh, M.E., Al-Hasani, S., Stieber, M. et al. (1996) The effect of chromatin condensation (aniline blue staining) and morphology (strict criteria) of human spermatozoa on fertilization, cleavage and pregnancy rates in an intracytoplasmic sperm injection program. Hum. Reprod., 11, 2468–2471.
Hammadeh, M.E., Stieber, M., Haidl, G. et al. (1997) Sperm count in ejaculates and after sperm selection with discontinuous percoll gradient centrifugation technique, as a prognostic index of IVF outcome. Archs Gynecol. Obstet., 259, 125–131.
Hofmann, N. and Hilscher, B. (1991) Use of aniline blue to assess chromatin condensation in morphologically normal spermatozoa in normal and infertile men. Hum. Reprod., 6, 979–982.
Hoshi, K., Katayose, H., Yanagida, K. et al. (1996) The relationship between acridine orange fluorescence of sperm nuclei and the fertilization ability of human sperm. Fertil. Steril., 66, 634–639.[Web of Science][Medline]
Ibrahim, M.E. and Pedersen, H. (1988) Acridine orange fluorescence as male fertility test. Arch. Androl., 20, 125–129[Web of Science][Medline]
Irvine, D.S., Twigg, J.P., Gordon, E.L. et al. (2000) DNA integrity in human spermatozoa: relationships with semen quality. J. Androl., 21, 33–34.[Abstract]
Janny, L. and Ménézo, Y.J. (1994) Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol. Reprod. Dev., 38, 36–42.[Web of Science][Medline]
Kruger, T.F., Menkveld, R., Stander, F.S. et al. (1986) Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil. Steril., 46, 1118–1123.[Web of Science][Medline]
Larson, K.L., Brannian, J.D., Timm, B.K. et al. (1999) Density gradient centrifugation and glass wool filtration of semen remove sperm with damaged chromatin structure. Hum. Reprod., 14, 2015–2019.
Liu, J., Nagy, Z., Joris, H. et al. (1994) Intracytoplasmic sperm injection does not require special treatment of the spermatozoa. Hum. Reprod., 9, 1127–1130.
Lopes, S., Sun, J.G., Jurisicova, A., Meriano, J. et al. (1998) Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil. Steril., 69, 528–532.[Web of Science][Medline]
Moosani, N., Pattinson, H.A., Carter, M.D. et al. (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil. Steril., 64, 811–817.[Web of Science][Medline]
Oehninger, S. (1996) Intracytoplasmic sperm injection: results from Norfolk, USA. Hum. Reprod., 11, 73–75.[Medline]
Palermo, G., Joris, H., Devroey, P. et al. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 17–18.[Web of Science][Medline]
Parinaud, J., Mieusset, R., Vieitez, G. et al. (1993) Influence of sperm parameters on embryo quality. Fertil. Steril., 60, 888–892.[Web of Science][Medline]
Parinaud, J., LeLannou, D., Vieitez, G. et al. (1997) Enhancement of motility by treating spermatozoa with an antioxidant solution (Sperm-Fit) following ejaculation. Hum. Reprod., 12, 2434–2436.
Roy, S.K., Kurz, S.G., Carlson, A.M. et al. (1998) Transforming growth factor ß receptor expression in hyperstimulated human granulosa cells and cleavage potential of the zygotes. Biol. Reprod., 59, 1311–1316.
Sailer, B.L., Jost, L.K., Evenson, D.P. (1995) Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J. Androl., 16, 80–87.
Sakkas, D. (1999) The use of blastocyst culture to avoid inheritance of an abnormal paternal genome after ICSI. Hum. Reprod., 14, 4–5.
Sanchez, R., Stalf, T., Khanaga, O. et al. (1996) Sperm selection methods for intracytoplasmic sperm injection (ICSI) and andrological patients. Andrology, 13, 228–233.
SAS Institute Inc. (1988) SAS/STAT[nabla] Users Guide, Release 6.03 Edition. SAS Institute Inc., Cary, North Carolina.
Shoukir, Y., Chardonnens, D., Campana, A. et al. (1998) Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence? Hum. Reprod., 13, 1632–1637.
Spano, M., Bonde, J.P., Hjollund, H.I. et al. (2000) Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil. Steril., 73, 43–50.[Web of Science][Medline]
Sukcharoen, N., Keith, J., Irvine, D.S. et al. (1995) Predicting the fertilizing potential of human sperm suspensions in vitro: importance of sperm morphology and leukocyte contamination. Fertil. Steril., 63, 1293–1300.[Web of Science][Medline]
Twigg, J.P., Irvine, D.S. and Aitken, R.J. (1998) Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum. Reprod., 13, 1864–1871.
Vanderzwalmen, P., Bertin Segal, G., Geerts, L. et al. (1991) Sperm morphology and IVF pregnancy rate: comparison between Percoll gradient centrifugation and swim-up procedures. Hum. Reprod., 6, 581–588.
Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993) High fertilization rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 1061–1066.
Vawda, A.I., Gunby, J. and Younglai, E.V. (1996) Semen parameters as predictors of in-vitro fertilization: the importance of strict criteria sperm morphology. Hum. Reprod., 11, 1445–1450.
Veeck, L.L. (1986) Atlas of the Human Oocyte and Early Conceptus, Vol. 1. Williams and Wilkins, Baltimore.
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. Cambridge University Press, Cambridge.
Submitted on November 5, 1999; accepted on April 26, 2000.
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D.Y. Liu and H.W.G. Baker Human sperm bound to the zona pellucida have normal nuclear chromatin as assessed by acridine orange fluorescence Hum. Reprod., June 1, 2007; 22(6): 1597 - 1602. [Abstract] [Full Text] [PDF]
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 C. Yildiz, P. Ottaviani, N. Law, R. Ayearst, L. Liu, and C. McKerlie Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse Reproduction, March 1, 2007; 133(3): 585 - 595. [Abstract] [Full Text] [PDF]
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M. Bungum, P. Humaidan, A. Axmon, M. Spano, L. Bungum, J. Erenpreiss, and A. Giwercman Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome Hum. Reprod., January 1, 2007; 22(1): 174 - 179. [Abstract] [Full Text] [PDF]
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T.E. Schmid, B. Eskenazi, A. Baumgartner, F. Marchetti, S. Young, R. Weldon, D. Anderson, and A.J. Wyrobek The effects of male age on sperm DNA damage in healthy non-smokers Hum. Reprod., January 1, 2007; 22(1): 180 - 187. [Abstract] [Full Text] [PDF]
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O. Stahl, J. Eberhard, K. Jepson, M. Spano, M. Cwikiel, E. Cavallin-Stahl, and A. Giwercman Sperm DNA integrity in testicular cancer patients Hum. Reprod., December 1, 2006; 21(12): 3199 - 3205. [Abstract] [Full Text] [PDF]
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A. Borini, N. Tarozzi, D. Bizzaro, M.A. Bonu, L. Fava, C. Flamigni, and G. Coticchio Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART Hum. Reprod., November 1, 2006; 21(11): 2876 - 2881. [Abstract] [Full Text] [PDF]
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 A. J. Wyrobek, B. Eskenazi, S. Young, N. Arnheim, I. Tiemann-Boege, E. W. Jabs, R. L. Glaser, F. S. Pearson, and D. Evenson Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm PNAS, June 20, 2006; 103(25): 9601 - 9606. [Abstract] [Full Text] [PDF]
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G. B. Boe-Hansen, J. Fedder, A. K. Ersboll, and P. Christensen The sperm chromatin structure assay as a diagnostic tool in the human fertility clinic Hum. Reprod., June 1, 2006; 21(6): 1576 - 1582. [Abstract] [Full Text] [PDF]
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A. A. Makhlouf and C. Niederberger DNA Integrity Tests in Clinical Practice: It Is Not a Simple Matter of Black and White (or Red and Green) J Androl, May 1, 2006; 27(3): 316 - 323. [Full Text] [PDF]
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R. Smith, H. Kaune, D. Parodi, M. Madariaga, R. Rios, I. Morales, and A. Castro Increased sperm DNA damage in patients with varicocele: relationship with seminal oxidative stress Hum. Reprod., April 1, 2006; 21(4): 986 - 993. [Abstract] [Full Text] [PDF]
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D. Evenson Reply to: 'The predictive value of the sperm chromatin structure assay (SCSA)'--a response from the SCSA inventor. Hum. Reprod., February 1, 2006; 21(2): 570 - 572. [Full Text] [PDF]
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M. Bungum, L. Bungum, and P. Humaidan A prospective study, using sibling oocytes, examining the effect of 30 seconds versus 90 minutes gamete co-incubation in IVF Hum. Reprod., February 1, 2006; 21(2): 518 - 523. [Abstract] [Full Text] [PDF]
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K. R. Chohan, J. T. Griffin, M. Lafromboise, C. J. De Jonge, and D. T. Carrell Comparison of Chromatin Assays for DNA Fragmentation Evaluation in Human Sperm J Androl, January 1, 2006; 27(1): 53 - 59. [Abstract] [Full Text] [PDF]
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V. W. Aoki, S. I. Moskovtsev, J. Willis, L. Liu, J. B. M. Mullen, and D. T. Carrell DNA Integrity Is Compromised in Protamine-Deficient Human Sperm J Androl, November 1, 2005; 26(6): 741 - 748. [Abstract] [Full Text] [PDF]
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J. Rubes, S. G. Selevan, D. P. Evenson, D. Zudova, M. Vozdova, Z. Zudova, W. A. Robbins, and S. D. Perreault Episodic air pollution is associated with increased DNA fragmentation in human sperm without other changes in semen quality Hum. Reprod., October 1, 2005; 20(10): 2776 - 2783. [Abstract] [Full Text] [PDF]
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E. Greco, S. Romano, M. Iacobelli, S. Ferrero, E. Baroni, M. G. Minasi, F. Ubaldi, L. Rienzi, and J. Tesarik ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment Hum. Reprod., September 1, 2005; 20(9): 2590 - 2594. [Abstract] [Full Text] [PDF]
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R.-M. Laberge and G. Boissonneault On the Nature and Origin of DNA Strand Breaks in Elongating Spermatids Biol Reprod, August 1, 2005; 73(2): 289 - 296. [Abstract] [Full Text] [PDF]
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E. W. Silver, B. Eskenazi, D. P. Evenson, G. Block, S. Young, and A. J. Wyrobek Effect of Antioxidant Intake on Sperm Chromatin Stability in Healthy Nonsmoking Men J Androl, July 1, 2005; 26(4): 550 - 556. [Abstract] [Full Text] [PDF]
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 E. Seli and D. Sakkas Spermatozoal nuclear determinants of reproductive outcome: implications for ART Hum. Reprod. Update, July 1, 2005; 11(4): 337 - 349. [Abstract] [Full Text] [PDF]
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E. Greco, M. Iacobelli, L. Rienzi, F. Ubaldi, S. Ferrero, and J. Tesarik Reduction of the Incidence of Sperm DNA Fragmentation by Oral Antioxidant Treatment J Androl, May 1, 2005; 26(3): 349 - 353. [Abstract] [Full Text] [PDF]
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S. Banks, S. A King, D S. Irvine, and P. T K Saunders Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa Reproduction, April 1, 2005; 129(4): 505 - 514. [Abstract] [Full Text] [PDF]
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J. Nicopoullos, C. Gilling-Smith, P. Almeida, and J. Ramsay The predictive value of sperm chromatin structure assay Hum. Reprod., March 1, 2005; 20(3): 839 - 839. [Full Text] [PDF]
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J. H. Check and R. W. Johnson Support for the contention that sperm with abnormal sperm chromatin structure assays are associated with reduced embryo implantation potential Hum. Reprod., March 1, 2005; 20(3): 840 - 841. [Full Text] [PDF]
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A. Berkovitz, F. Eltes, S. Yaari, N. Katz, I. Barr, A. Fishman, and B. Bartoov The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm Hum. Reprod., January 1, 2005; 20(1): 185 - 190. [Abstract] [Full Text] [PDF]
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E. Greco, F. Scarselli, M. Iacobelli, L. Rienzi, F. Ubaldi, S. Ferrero, G. Franco, N. Anniballo, C. Mendoza, and J. Tesarik Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa Hum. Reprod., January 1, 2005; 20(1): 226 - 230. [Abstract] [Full Text] [PDF]
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U. Paasch, R. K. Sharma, A. K. Gupta, S. Grunewald, E. J. Mascha, A. J. Thomas Jr, H.-J. Glander, and A. Agarwal Cryopreservation and Thawing Is Associated with Varying Extent of Activation of Apoptotic Machinery in Subsets of Ejaculated Human Spermatozoa Biol Reprod, December 1, 2004; 71(6): 1828 - 1837. [Abstract] [Full Text] [PDF]
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J. Erenpreiss, K. Jepson, A. Giwercman, I. Tsarev, Je. Erenpreisa, and M. Spano Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays Hum. Reprod., October 1, 2004; 19(10): 2277 - 2282. [Abstract] [Full Text] [PDF]
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ESHRE Capri Workshop Group Diagnosis and management of the infertile couple: missing information Hum. Reprod. Update, July 1, 2004; 10(4): 295 - 307. [Abstract] [Full Text] [PDF]
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M. Bungum, P. Humaidan, M. Spano, K. Jepson, L. Bungum, and A. Giwercman The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI Hum. Reprod., June 1, 2004; 19(6): 1401 - 1408. [Abstract] [Full Text] [PDF]
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L. Gandini, F. Lombardo, D. Paoli, F. Caruso, P. Eleuteri, G. Leter, R. Ciriminna, F. Culasso, F. Dondero, A. Lenzi, et al. Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage Hum. Reprod., June 1, 2004; 19(6): 1409 - 1417. [Abstract] [Full Text] [PDF]
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A. M. Codrington, B. F. Hales, and B. Robaire Spermiogenic Germ Cell Phase-Specific DNA Damage Following Cyclophosphamide Exposure J Androl, May 1, 2004; 25(3): 354 - 362. [Abstract] [Full Text] [PDF]
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 L. Muriel, E. Segrelles, V. Goyanes, J. Gosalvez, and J. L. Fernandez Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis Mol. Hum. Reprod., March 1, 2004; 10(3): 203 - 209. [Abstract] [Full Text] [PDF]
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J. Tesarik, E. Greco, and C. Mendoza Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation Hum. Reprod., March 1, 2004; 19(3): 611 - 615. [Abstract] [Full Text] [PDF]
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M. H. Moustafa, R. K. Sharma, J. Thornton, E. Mascha, M. A. Abdel-Hafez, A. J. Thomas, and A. Agarwal Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility Hum. Reprod., January 1, 2004; 19(1): 129 - 138. [Abstract] [Full Text] [PDF]
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R. Hauser, N.P. Singh, Z. Chen, L. Pothier, and L. Altshul Lack of an association between environmental exposure to polychlorinated biphenyls and p,p'-DDE and DNA damage in human sperm measured using the neutral comet assay Hum. Reprod., December 1, 2003; 18(12): 2525 - 2533. [Abstract] [Full Text] [PDF]
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S. Loft, T. Kold-Jensen, N. H. Hjollund, A. Giwercman, J. Gyllemborg, E. Ernst, J. Olsen, T. Scheike, H. E. Poulsen, and J. P. Bonde Oxidative DNA damage in human sperm influences time to pregnancy Hum. Reprod., June 1, 2003; 18(6): 1265 - 1272. [Abstract] [Full Text] [PDF]
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M. Benchaib, V. Braun, J. Lornage, S. Hadj, B. Salle, H. Lejeune, and J. F. Guerin Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique Hum. Reprod., May 1, 2003; 18(5): 1023 - 1028. [Abstract] [Full Text] [PDF]
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J. L. Fernandez, L. Muriel, M. T. Rivero, V. Goyanes, R. Vazquez, and J. G. Alvarez The Sperm Chromatin Dispersion Test: A Simple Method for the Determination of Sperm DNA Fragmentation J Androl, January 1, 2003; 24(1): 59 - 66. [Abstract] [Full Text] [PDF]
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E.H. Duran, M. Morshedi, S. Taylor, and S. Oehninger Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study Hum. Reprod., December 1, 2002; 17(12): 3122 - 3128. [Abstract] [Full Text] [PDF]
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J. Richthoff, M. Spano, Y.L. Giwercman, B. Frohm, K. Jepson, J. Malm, S. Elzanaty, M. Stridsberg, and A. Giwercman The impact of testicular and accessory sex gland function on sperm chromatin integrity as assessed by the sperm chromatin structure assay (SCSA) Hum. Reprod., December 1, 2002; 17(12): 3162 - 3169. [Abstract] [Full Text] [PDF]
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J. Erenpreiss, S. Hlevicka, J. Zalkalns, and J. Erenpreisa Effect of Leukocytospermia on Sperm DNA Integrity: A Negative Effect in Abnormal Semen Samples J Androl, September 1, 2002; 23(5): 717 - 723. [Abstract] [Full Text] [PDF]
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M. Tomsu, V. Sharma, and D. Miller Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters Hum. Reprod., July 1, 2002; 17(7): 1856 - 1862. [Abstract] [Full Text] [PDF]
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I.D. Morris, S. Ilott, L. Dixon, and D.R. Brison The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development Hum. Reprod., April 1, 2002; 17(4): 990 - 998. [Abstract] [Full Text] [PDF]
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K. Lundin, C. Bergh, and T. Hardarson Early embryo cleavage is a strong indicator of embryo quality in human IVF Hum. Reprod., December 1, 2001; 16(12): 2652 - 2657. [Abstract] [Full Text] [PDF]
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M.J. Tomlinson, O. Moffatt, G.C. Manicardi, D. Bizzaro, M. Afnan, and D. Sakkas Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception Hum. Reprod., October 1, 2001; 16(10): 2160 - 2165. [Abstract] [Full Text] [PDF]
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M. Ollero, E. Gil-Guzman, M. C. Lopez, R. K. Sharma, A. Agarwal, K. Larson, D. Evenson, A. J. Thomas Jr, and J. G. Alvarez Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility Hum. Reprod., September 1, 2001; 16(9): 1912 - 1921. [Abstract] [Full Text] [PDF]
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U. Zollner, K.-P. Zollner, J. Dietl, and T. Steck Semen sample collection in medium enhances the implantation rate following ICSI in patients with severe oligoasthenoteratozoospermia Hum. Reprod., June 1, 2001; 16(6): 1110 - 1114. [Abstract] [Full Text] [PDF]
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