- Email: [email protected]
Adequate ovarian follicular status does not prevent the decrease in pregnancy rates associated with high sperm DNA fragmentation
Adequate ovarian follicular status does not prevent the decrease in pregnancy rates associated with high sperm DNA fragmentation Nelly Frydman, Pharm.D.,a,d Nadia Prisant, M.D.,a Laetitia Hesters, Pharm.D.,a,d Ren e Frydman, Ph.D.,b,d G erard Tachdjian, Ph.D.,a,d Paul Cohen-Bacrie, Pharm.D.,c and R enato Fanchin, Ph.D.b,d a Department of Genetics and Reproduction, and b Department of Gynecology and Obstetrics, Antoine Beclere Hospital, Clamart; c Laboratoire d’Eylau, Paris; and d INSERM, U782, Clamart, France
Objective: Potential reparation of sperm DNA fragmentation in the oocyte may disturb any relationship between DNA-damaged sperm and the implantation ability of resulting embryos. To rule out this factor, we analyzed the consequences of sperm DNA fragmentation on IVF-ET outcome in women with healthy ovarian function. Design: Prospective study. Setting: Teaching hospital, France. Patient(s): All 117 women were <38 years old, who combined normal serum day-3 FSH and inhibin B levels with an adequate response to controlled ovarian hyperstimulation. Intervention(s): The DNA fragmentation rate was determined in the raw sperm used for conventional IVF by flow cytometric terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assay. Cycles were sorted into two groups according to whether DNA fragmentation exceeded (high fragmentation [HF], n ¼ 52) or did not exceed (low fragmentation [LF], n ¼ 65) the 50th percentile of values (35%). Main Outcome Measure(s): D2 embryo quality and implantation and ongoing pregnancy rates. Result(s): Patients’ characteristics, raw semen parameters, fertilization rates, and embryology data were similar in HF and LF groups. Clinical (37.5% vs. 62.5%) and ongoing (23.5% vs. 57.8%) pregnancy rates per ET and implantation rates (24.5% vs. 42.4%) were lower in the HF group than in the LF group. Conclusion(s): High sperm DNA fragmentation spares fertilization and top embryo morphology rates but is associated with decreased IVF-ET outcome. (Fertil Steril 2008;89:92–7. 2008 by American Society for Reproductive Medicine.) Key Words: Conventional IVF, ongoing pregnancy rate, sperm DNA fragmentation, TUNEL assay
More than 120,000 conventional IVF-ET cycles were performed in Europe in 2001 (1). Male factor infertility contributes to roughly 50% of cases (2). Yet routine semen parameters are considered to be poor prognosticators of treatment outcome, at least in part because they fail to detect DNA damage (3). To overcome this limitation of male infertility explorations, different types of assays, with comparable reliability, have been developed to directly assess the extent of sperm DNA damage (4): 1) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay, which quantifies by flow cytometry, fluorescent microscopy, or light microscopy the incorporation of labeled dUTP into single- and doublestranded DNA breaks (5–7); 2) Comet assay, which quantifies single- and doublestranded breaks associated with DNA damage through increased comet tail fluorescent intensity and length (8–11); Received September 26, 2006; revised and accepted February 12, 2007. ne tique et ReproducReprint requests: Dr. Nelly Frydman, Service de Ge cle re, INSERM U782, Universite Paris 11, 157, tion, Hoˆpital Antoine Be rue de la porte de Trivaux 92140 Clamart, France (FAX 00 33 1 45374207; E-mail: [email protected]).
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3) Flow cytometric sperm chromatin structure assay (SCSA), which assesses damaged sperm chromatin susceptibility to physical denaturation in situ resulting in metachromatic shift from green (double-stranded nucleic acid) to red fluorescence (single-stranded nucleic acid) (12); and 4) Sperm chromatin dispersion (SCD) test, which is based on an induced decondensation (13). These diagnostic techniques have been instrumental in evaluating the possible functional consequences of sperm DNA fragmentation. However, whereas some investigators show that motility and morphology of DNA-damaged sperm may remain unaffected (14), the ability of such a sperm to adequately fertilize the oocyte (15–17) and generate a morphologically normal embryo (5, 15, 16, 18) and viable pregnancies (16, 19, 20) remains a matter of debate. However, a possible explanation for these controversial results is the reported ability of healthy oocytes to repair DNA-damaged spermatozoa, as shown in rodents (21, 22). Unfortunately, in the earlier studies (15–20) the ovarian status data were not available. Therefore, we hypothesized that DNA-repairing ability of the oocyte may exert a confounding effect in the assessment
Fertility and Sterility Vol. 89, No. 1, January 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
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of embryologic effects of sperm DNA fragmentation. An attractive model to rule out this bias might be the study of IVF-ET candidates endowed with healthy ovarian function. It is conceivable that, in this group of patients, DNA-repairing oocytes are more frequent than in ovarian-aged patients, and the adverse effects of sperm DNA fragmentation on IVF-ET outcome may be null or attenuated.
According to usual sperm parameters, we performed conventional IVF using 5,000 to 10,000 progressive typical spermatozoa for one cumulus oocyte complex, in 35-mL microdrops of IVF medium (MediCult, Lyon, France) under paraffin oil (MediCult) at 37 C under an atmosphere of 5% CO2. Zygotes were cultured in microdrops of 35 mL ISM1 culture medium (MediCult) until day 2.
To address this issue, we analyzed the consequences of sperm DNA fragmentation on oocyte fertilization, embryology, and embryo implantation outcome in selected IVF-ET candidates having adequate ovarian follicular status.
Embryo morphology was then graded as A, B, C, and D. Embryos with 4 blastomeres of similar size and no fragmentation were scored A. Those with 4 blastomeres of similar size and 10% to 20% fragmentation, or showing appropriate developmental stage (3 or 5 blastomeres), were scored B. Embryos with 20% to 30% fragmentation were labeled grade C, whatever the number of blastomeres. Those having more than 30% fragmentation and those with at least one multinucleated blastomere were scored D. Grade A and B embryos were considered ‘‘top-quality embryos.’’
MATERIAL AND METHODS Patients We prospectively studied 117 IVF-ET candidates. All women met the following inclusion criteria: 1) <38 years of age; 2) adequate ovarian follicular status, defined as normal serum FSH and inhibin B levels measured on cycle day 3 (<10 mIU/mL and >45 pg/mL, respectively) within the 6 months preceding IVF-ET; and 3) adequate response to controlled ovarian hyperstimulation (COH) defined as mature follicle (R16 mm) count on the day of hCG administration of R5. In addition, sperm parameters had to be suitable for a classic IVF-ET (>1 106 of progressive spermatozoa obtained after sperm migration). Indications for IVF-ET were tubal factor (59%), grade I endometriosis (12%), or unexplained infertility (29%). Informed consent was obtained from all couples, and this investigation received the approval of our internal Institutional Review Board. Controlled Ovarian Hyperstimulation Protocol As described elsewhere (23), women received a GnRH agonist (Decapeptyl; Beaufour Ipsen Pharma, Paris, France) on cycle day 2. After a confirmed complete pituitary desensitization, hMG therapy was initiated (Menopur; Ferring Pharmaceuticals, Gentilly, France). Administration of hCG (Gonadotrophine Chorionique ‘‘Endo’’; Organon Pharmaceuticals, Saint-Denis, France) was performed when at least 5 follicles exceeded 16 mm in diameter. Oocyte retrieval (OR) was performed approximately 36 hours after hCG administration by transvaginal ultrasound–guided aspiration.
Embryo transfers were performed using a classic Frydman catheter (CCD, Paris, France). In all patients, luteal phase was supported with micronized progesterone (600 mg/day Estima; Effik Pharmaceuticals, Bievres, France) administered daily by vaginal route starting on the evening of ET. Measurement of DNA Fragmentation by TUNEL Assay and Flow Cytometry From the ejaculate eventually used for IVF in each patient, a sperm aliquot (300–500 mL) was constituted to further DNA fragmentation assessment by the TUNEL assay (5– 7). Each of them contained at least 2 106 spermatozoa, which was diluted in 0.5 mL of phosphate-buffered saline (PBS) solution and then fixed by the addition of 1 mL 1% formaldehyde (Sigma Chemical Co., St. Louis, MO) for 60 minutes at room temperature. After 10-minute centrifugation, the pellet was resuspended in PBS and centrifuged again for 10 minutes. Afterwards, 10 mL sodium citrate 1% (MSD, Paris, France) with Triton X-100 (final concentration 0.1%; Supelco, Paris, France) was added to permeabilize cell membranes. After two more washes in PBS, the fixed permeabilized sperm was kept at 4 C in the dark until analysis.
Sperm Preparation, Fertilization, and Embryo Culture Sperm samples were collected by masturbation. Samples were analyzed for total sperm number, concentration, motility, and morphology. Sperm selection was done by migration on a PureSperm gradient (Nidacon; JCD, Lyon, France). Sperm was layered upon a 45%:90% PureSperm density gradient, processed by centrifuge at 600g for 15 minutes, and resuspended in 1 mL capacitating medium (FerticultFertipro; JCD).
For the assessment of DNA fragmentation, the final preparation was centrifuged for 10 minutes and the pellet diluted in 50 mL staining buffer containing 3mmol/L biotine 16dUTP and 10 U terminal deoxynucleotidyl transferase (TdT; Roche Diagnostics, Meylan, France) and incubated at 37 C for 45 minutes. Spermatozoa were then treated with streptavidin-fluorescein (Roche Diagnostics) following by incubation at room temperature in the dark for 40 minutes. Stained spermatozoa were washed twice in PBS and rediluted in 750 mL PBS containing 10 mg/mL propidium iodide (Sigma Chemical Co.). Negative control sperm cells were treated identically except for the omission of enzymatic solution. Positive control sperm cells were treated identically after exposition to DNAse I (Roche Diagnostics) for 30 minutes.
After density-gradient separation, a second evaluation of concentration, motility, and morphology was performed.
Sperm DNA fragmentation and propidium iodide labeling were analyzed with a flow cytometer (EPICS-XL6MCL;
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Beckman Coulter, Villepinte, France) equipped with a 15 mW argon-ion laser as the light source. The flow rate was set at approximately 500 spermatozoa/s, and 5,000 to 10,000 stained spermatozoa were analyzed in each sample. Light-scatter and fluorescence data were obtained at a fixed gain setting in logarithmic mode. The green fluorescence (FL1) signals were detected through a 530 nm band pass filter, and red fluorescence (PI) signals were collected through a 620 nm band pass filter. To distinguish spermatozoa from debris, a dot plot distribution of spermatozoa, based on linear forward scatter, which correlates with cell size, and side scatter, which correlates with cell density, was used. The data were processed on a computer using Lysis software. The percentage of DNA-fragmented sperm in each sample was determined by subtracting negative control green fluorescence histograms from TdT-positive ones. Definition of Sperm DNA Fragmentation Groups According to the percentage of DNA-fragmented sperm remaining inferior to or exceeding 35%, 2 study groups were determined: low fragmentation group (<35%, LF, n ¼ 65) and high fragmentation group (R35%, HF, n ¼ 52). The choice of 35% threshold was arbitrary and based on the fact that it corresponds to the rounded median value (50th percentile) of DNA fragmentation rate in the present population. Statistics The measure of central tendency used was the mean, and the measure of variability was the standard deviation. The 95% confidence intervals (CIs) were estimated when necessary.
When normality of data could not be ascertained, medians and minimum-maximum values were used. Differences of continuous variables in LF and HF groups were analyzed using the Student t test and those of proportions with the chi-squared test. The Mann-Whitney test was used for nonparametric data. When the P value was < .05, the difference was considered to be statistically significant. RESULTS Patients’ Characteristics and Ovarian Response to COH Patients’ characteristics in LF and HF groups are detailed in Table 1. As shown, female age, day 3 ovarian reserve assessment (serum FSH and inhibin B levels), rank of the current IVF-ET attempt, and indications for IVF-ET were similar in LF and HF groups. In addition, characteristics of the ovarian response to COH were similar in both groups. Sperm Characteristics Overall, median sperm characteristics met the usual criteria needed to perform conventional IVF-ET. Sperm concentration before PureSperm preparation was 20 (range 7–260) 106 spermatozoa/mL and after was 20 (range 7–276) 106 spermatozoa/mL; sperm motility before preparation was 40% (range 20%–60%) and after was 90% (range 60%–95%). As shown in Table 2, both groups (LF and HF) were comparable regarding semen parameters. Embryology Data and IVF-ET Outcome Embryology data and IVF-ET outcome in LF and HF groups are summarized in Table 3. Both groups were comparable regarding the total number of retrieved and inseminated oocytes, fertilization rates, and number of embryos obtained.
TABLE 1 Female population characteristics in the low (LF) and high (HF) fragmentation groups.
Age of women (yrs)a Basal FSH (mUI/mL)a Inhibin B (pg/mL)a Rank of IVF-ET attempt Tubal factor Grade I endometriosis Unexplained hMG requirement (UI) Day of hCG administration E2 level (pg/mL)b Number of follicles R16 mma,b Endometrial thickness (mm)b Number of ET
LF (n [ 65)
HF (n [ 52)
P value
33.1 [23–37] 5.9 [2.3–9.9] 77 [45–185] 2.02 1.5 37 (57%) 7 (11%) 21 (32%) 2477 801 11.9 0.2 3066 1160 8 [5–13] 9.8 0.2 64
33.3 [22–37] 6.2 [2.6–9.9] 74 [45–180] 1.71 1.03 32 (62%) 7 (13%) 13 (25%) 2621 819 11.9 0.2 2845 1130 7.5 [5–15] 9.7 0.3 51
NS NS NS NS NS NS NS NS NS NS NS NS —
Note: NS ¼ not significant. a Median [range]. b On the day of hCG administration. Frydman. Sperm DNA fragmentation and ART outcome. Fertil Steril 2008.
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In line with this, the prevalence of top embryos obtained, the number of transfered embryos, and the prevalence of topquality embryos transfered were similar in LF and HF groups. As also shown in Table 3, clinical pregnancy rates (gestational sac with positive heartbeat observed ultrasonographically at 7 weeks of amenorrhoea), ongoing pregnancy rates (>12 weeks of amenorrhoea) per OR, and implantation rates (total number of gestational sacs 100/total number of embryos transfered) were statistically higher in the LF group than in the HF group. Furthermore, miscarriage rates were statistically decreased in the LF compared with the HF group. DISCUSSION The present clinical investigation represents an attempt to test the hypothesis that healthy oocytes could be able to repair sperm DNA fragmentation (21, 22) and compensate for its reported adverse effects on IVF-ET results (10, 12, 24). Indeed, it is possible that a number of physiologic sperm functions are compromised by chromatin fragmentation at, at least, two levels. On the one hand, sperm DNA fragmentation may hinder some putative early paternal effects (6), such as adequate oocyte fertilization, embryo morphology, and implantation (5, 18, 25). On the other hand, late paternal functions may be altered by this condition as the sperm aptitude to generate embryos leading to viable pregnancies (6, 15, 17, 19). Yet, women endowed with adequate ovarian function, and presumably more likely to produce DNA-repairing oocytes, should display unaltered IVF-ET results despite increased sperm DNA fragmentation rates. In such a selected female population, we observed that oocyte fertilization rates and early embryo morphology remained unaltered. Yet, this lack of detrimental effect of high sperm DNA fragmentation
may not be exclusively attributed to the DNA-repairing aptitude, if any, of oocytes. Indeed, because the paternal genome is barely expressed before the third cycle of embryo cell division (26), it is unlikely that sperm DNA fragmentation could encumber oocyte fertilization or early embryo characteristics. Yet the issue of whether sperm DNA damage may affect later stages of blastomere division could not be addressed by the present study and remains to be investigated. A trend for reduced blastocyst development rate has been noticed in the presence of high sperm DNA fragmentation (exceeding 20%) in a preliminary report (25). Incidentally, those authors assessed the extent of DNA fragmentation by TUNEL assay in an unselected female population (25). In addition, even in women displaying healthy ovarian function, we observed significantly lower clinical pregnancy and embryo implantation rates in the HF group than in the LF group. A number of hypotheses may be considered to explain this result. First, it is conceivable that patient selection based merely on clinical criteria of adequate ovarian functioning was insufficient to thoroughly exclude oocytes with deficient DNA-repairing competence. Unfortunately, by design, adequate oocyte morphology could not be retained as an additional criterion for patient selection in the present investigation, because only cases of conventional IVF-ET were included. Yet, it is noteworthy that reliable criteria for identifying oocytes that are more prone to repair sperm DNA fragmentation are as yet not available. Second, it is possible that the oocyte, irrespective of its quality, is inherently unable to overcome unwanted effects of extensive sperm DNA damage on embryo viability. This pivotal issue deserves to be the matter of further basic and
TABLE 2 Sperm characteristics before and after preparation in the low (LF) and high (HF) fragmentation groups.
Before sperm preparationa Volume of semen Sperm concentration (106/mL) Vitality (%) Motility (% of global motility) Progressive motility (% of a þ b) Percentage of typical forms After sperm preparationa No. of progressive spermatozoa (106) Motility (% of global motility) Progressive motility (% of a þ b)
LF (n [ 65)
HF (n [ 52)
P value
3.5 [1.5–7.0] 17.1 [7–260] 70 [51–95] 40 [30–60] 30 [20–50] 46 [19–67]
3.5 [1.5–7.0] 17.8 [7–150] 70 [55–90] 40 [20–60] 30 [10–50] 40 [19–62]
NS NS NS NS NS NS
15 [1–112] 90 [70–95] 80 [60–90]
18 [2–84] 90 [60–95] 80 [50–90]
NS NS NS
Note: NS ¼ not significant. a Median [range]. Frydman. Sperm DNA fragmentation and ART outcome. Fertil Steril 2008.
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TABLE 3 Embryology data and IVF-ET outcome in the low (LF) and high (HF) fragmentation groups.
No. of oocytes retrieved No. of 2 pro-nuclei zygotes No. of 3 pro-nuclei zygotes Fertilization rate [95% CI] No. of embryos obtained Prevalence of top-quality embryos [95% CI] No. of transfered embryos Prevalence of top-quality embryos transfered [95% CI] Clinical pregnancy rate per OR [95% CI] Implantation rate [95% CI] Ongoing pregnancy rate per OR [95% CI] Miscarriage rate [95% CI] Live birth rate per OR [95% CI] Take-home babies
LF (n [ 65)
HF (n [ 52)
P value
11.7 0.6 7.0 0.5 1.0 0.1 69.9% [64.4–75.3] 7.9 0.5 62.2% [54.9–69.5] 2.1 0.06 93.9% [88.5–99.3] 62.5% [50.4–74.6] 42.4% [33.8–51.0] 57.8% [45.5–70.1] 10.0% [0.51–19.5] 56.2% [43.8–68.6] 46
11.3 0.6 7.2 0.5 0.9 0.1 71.7% [66.0–77.4] 7.4 0.5 62.5% [54.4–70.6] 2.1 0.06 91.2% [84.6–97.8] 37.5% [24.0–51] 24.5% [16.0–33.0] 23.5% [11.7–35.3] 36.8% [14.7–58.9] 23.5% [11.7–35.3] 16
NS NS NS NS NS NS NS NS .007 .0043 .0002 .01 .0004
Note: CI ¼ confidence interval; NS ¼ not significant; OR ¼ oocyte retrieval. Frydman. Sperm DNA fragmentation and ART outcome. Fertil Steril 2008.
clinical research. Further, the poorer IVF-ET outcome associated with extensive sperm DNA fragmentation may result from alteration of the embryo potential to reach the blastocyst stage (20, 25), to successfully implant into the uterus, or both. Yet the present results are not contributive to clarifying these issues which deserve additional investigation.
characteristics. Also, the development of clinical and biologic measures aiming at repairing sperm DNA pathologies and/or selecting healthy spermatozoa before IVF-ET should be considered to improve spontaneous or assisted fertility.
The present data also showed a more than threefold increase in miscarriage rates in women included in the HF group compared with the LF group, in agreement with data from other investigators (19). This strongly suggests an involvement of sperm DNA fragmentation in the development competence of the implanted embryo (late paternal effect). However, the mechanisms underlying this phenomenon remain unclear. It may be due to ‘‘abortive apoptosis,’’ a pathologic condition known to yield the persistence of high level of sperm DNA fragmentation in raw semen (27–29).
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In support of this hypothesis, a positive relationship between percentage of apoptotic sperm, sperm aneuploidy level, and recurrent miscarriage has been reported (30). In agreement with this data, high sperm DNA fragmentation rates have also been associated with recurrent miscarriages (31). In conclusion, the present investigation shows that a healthy ovarian follicular status does not compensate for the putative adverse effects of increased sperm DNA fragmentation on embryo implantation and intrauterine embryo development potential. These results challenge but do not completely disprove the hypothesis that competent oocytes are able to repair any extent of sperm DNA damage and to reduce the detrimental effect of this condition on embryogenesis. Furthermore, the present study emphasizes that routine assessment of the extent of sperm DNA damage should be performed during infertility explorations irrespective of sperm 96
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1. The European IVF-monitoring programme (EIM), for the European Society of Human Reproduction and Embryology (ESHRE). Assisted reproductive technology in Europe, 2002. Results generated from European registers by ESHRE. Hum Reprod 2006;21:1680–97. 2. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15:1338–44. 3. Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003;9:331–45. 4. Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril 2001;4:674–7. 5. Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod 2003;18:1023–8. 6. Tesarik J, Greco E, Mendoza C. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Hum Reprod 2004;19:611–5. 7. Greco E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Tesarik J. Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J Androl 2005;26:349–53. 8. Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W. A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay. Mol Hum Reprod 1996;2:613–9. 9. Singh NP, Stephens RE. X-Ray induced DNA double-strand breaks in human sperm. Mutagenesis 1998;13:75–9. 10. Morris ID, Ilott S, Dixon L, Brison DR. The spectrum of DNA damage in human sperm assessed by single gel electrophoresis (comet assay) and its relationship to fertilization and embryo development. Hum Reprod 2002;17:990–8.
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11. Tomsu M, Sharma V, Miller D. Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Hum Reprod 2002;17:1856–62. 12. Larson KL, DeJonge CJ, Barnes AM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod 2000;15: 1717–22. 13. Muriel L, Meseguer M, Fernandez JL, Alvarez J, Remohi J, Pellicer A, et al. Value of the sperm chromatine dispersion test in predicting pregnancy outcome in intrauterine insemination: a blind prospective study. Hum Reprod 2006;21:738–44. 14. Saleh RA, Agarwal A, Nelson DR, Nada EA, El-Tonsy MH, Alvarez JG, et al. Increased sperm nuclear damage in normozoospermic infertile men: a prospective study. Fertil Steril 2002;78:313–8. 15. Filatov MV, Semenova EV, Vorob’eva OA, Leont’eva OA, Drobchenko EA. Relationship between abnormal sperm chromatin packing and IVF results. Mol Hum Reprod 1999;5:825–30. 16. Gandini L, Lombardo F, Paoli D, Caruso F, Eleuteri P, Leter G, et al. Fullterm pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Hum Reprod 2004;19:1409–17. 17. Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, et al. Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril 2004;81:965–72. 18. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602–7. 19. Borini A, Tarozzi N, Bizzaro D, Bonu MA, Fava L, Flamigni C, et al. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Hum Reprod 2006;21:2876–81. 20. Muriel L, Garrido N, Fernandez JL, Remohi J, Pellicer A, De los Santos MJ, et al. Value of the sperm deoxyribonucleic acid fragmentation level, as measured by sperm chromatin dispersion test, in outcome of in
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Objective: Potential reparation of sperm DNA fragmentation in the oocyte may disturb any relationship between DNA-damaged sperm and the implantation ability of resulting embryos. To rule out this factor, we analyzed the consequences of sperm DNA fragmentation on IVF-ET outcome in women with healthy ovarian function. Design: Prospective study. Setting: Teaching hospital, France. Patient(s): All 117 women were <38 years old, who combined normal serum day-3 FSH and inhibin B levels with an adequate response to controlled ovarian hyperstimulation. Intervention(s): The DNA fragmentation rate was determined in the raw sperm used for conventional IVF by flow cytometric terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assay. Cycles were sorted into two groups according to whether DNA fragmentation exceeded (high fragmentation [HF], n ¼ 52) or did not exceed (low fragmentation [LF], n ¼ 65) the 50th percentile of values (35%). Main Outcome Measure(s): D2 embryo quality and implantation and ongoing pregnancy rates. Result(s): Patients’ characteristics, raw semen parameters, fertilization rates, and embryology data were similar in HF and LF groups. Clinical (37.5% vs. 62.5%) and ongoing (23.5% vs. 57.8%) pregnancy rates per ET and implantation rates (24.5% vs. 42.4%) were lower in the HF group than in the LF group. Conclusion(s): High sperm DNA fragmentation spares fertilization and top embryo morphology rates but is associated with decreased IVF-ET outcome. (Fertil Steril 2008;89:92–7. 2008 by American Society for Reproductive Medicine.) Key Words: Conventional IVF, ongoing pregnancy rate, sperm DNA fragmentation, TUNEL assay
More than 120,000 conventional IVF-ET cycles were performed in Europe in 2001 (1). Male factor infertility contributes to roughly 50% of cases (2). Yet routine semen parameters are considered to be poor prognosticators of treatment outcome, at least in part because they fail to detect DNA damage (3). To overcome this limitation of male infertility explorations, different types of assays, with comparable reliability, have been developed to directly assess the extent of sperm DNA damage (4): 1) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay, which quantifies by flow cytometry, fluorescent microscopy, or light microscopy the incorporation of labeled dUTP into single- and doublestranded DNA breaks (5–7); 2) Comet assay, which quantifies single- and doublestranded breaks associated with DNA damage through increased comet tail fluorescent intensity and length (8–11); Received September 26, 2006; revised and accepted February 12, 2007. ne tique et ReproducReprint requests: Dr. Nelly Frydman, Service de Ge cle re, INSERM U782, Universite Paris 11, 157, tion, Hoˆpital Antoine Be rue de la porte de Trivaux 92140 Clamart, France (FAX 00 33 1 45374207; E-mail: [email protected]).
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3) Flow cytometric sperm chromatin structure assay (SCSA), which assesses damaged sperm chromatin susceptibility to physical denaturation in situ resulting in metachromatic shift from green (double-stranded nucleic acid) to red fluorescence (single-stranded nucleic acid) (12); and 4) Sperm chromatin dispersion (SCD) test, which is based on an induced decondensation (13). These diagnostic techniques have been instrumental in evaluating the possible functional consequences of sperm DNA fragmentation. However, whereas some investigators show that motility and morphology of DNA-damaged sperm may remain unaffected (14), the ability of such a sperm to adequately fertilize the oocyte (15–17) and generate a morphologically normal embryo (5, 15, 16, 18) and viable pregnancies (16, 19, 20) remains a matter of debate. However, a possible explanation for these controversial results is the reported ability of healthy oocytes to repair DNA-damaged spermatozoa, as shown in rodents (21, 22). Unfortunately, in the earlier studies (15–20) the ovarian status data were not available. Therefore, we hypothesized that DNA-repairing ability of the oocyte may exert a confounding effect in the assessment
Fertility and Sterility Vol. 89, No. 1, January 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
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of embryologic effects of sperm DNA fragmentation. An attractive model to rule out this bias might be the study of IVF-ET candidates endowed with healthy ovarian function. It is conceivable that, in this group of patients, DNA-repairing oocytes are more frequent than in ovarian-aged patients, and the adverse effects of sperm DNA fragmentation on IVF-ET outcome may be null or attenuated.
According to usual sperm parameters, we performed conventional IVF using 5,000 to 10,000 progressive typical spermatozoa for one cumulus oocyte complex, in 35-mL microdrops of IVF medium (MediCult, Lyon, France) under paraffin oil (MediCult) at 37 C under an atmosphere of 5% CO2. Zygotes were cultured in microdrops of 35 mL ISM1 culture medium (MediCult) until day 2.
To address this issue, we analyzed the consequences of sperm DNA fragmentation on oocyte fertilization, embryology, and embryo implantation outcome in selected IVF-ET candidates having adequate ovarian follicular status.
Embryo morphology was then graded as A, B, C, and D. Embryos with 4 blastomeres of similar size and no fragmentation were scored A. Those with 4 blastomeres of similar size and 10% to 20% fragmentation, or showing appropriate developmental stage (3 or 5 blastomeres), were scored B. Embryos with 20% to 30% fragmentation were labeled grade C, whatever the number of blastomeres. Those having more than 30% fragmentation and those with at least one multinucleated blastomere were scored D. Grade A and B embryos were considered ‘‘top-quality embryos.’’
MATERIAL AND METHODS Patients We prospectively studied 117 IVF-ET candidates. All women met the following inclusion criteria: 1) <38 years of age; 2) adequate ovarian follicular status, defined as normal serum FSH and inhibin B levels measured on cycle day 3 (<10 mIU/mL and >45 pg/mL, respectively) within the 6 months preceding IVF-ET; and 3) adequate response to controlled ovarian hyperstimulation (COH) defined as mature follicle (R16 mm) count on the day of hCG administration of R5. In addition, sperm parameters had to be suitable for a classic IVF-ET (>1 106 of progressive spermatozoa obtained after sperm migration). Indications for IVF-ET were tubal factor (59%), grade I endometriosis (12%), or unexplained infertility (29%). Informed consent was obtained from all couples, and this investigation received the approval of our internal Institutional Review Board. Controlled Ovarian Hyperstimulation Protocol As described elsewhere (23), women received a GnRH agonist (Decapeptyl; Beaufour Ipsen Pharma, Paris, France) on cycle day 2. After a confirmed complete pituitary desensitization, hMG therapy was initiated (Menopur; Ferring Pharmaceuticals, Gentilly, France). Administration of hCG (Gonadotrophine Chorionique ‘‘Endo’’; Organon Pharmaceuticals, Saint-Denis, France) was performed when at least 5 follicles exceeded 16 mm in diameter. Oocyte retrieval (OR) was performed approximately 36 hours after hCG administration by transvaginal ultrasound–guided aspiration.
Embryo transfers were performed using a classic Frydman catheter (CCD, Paris, France). In all patients, luteal phase was supported with micronized progesterone (600 mg/day Estima; Effik Pharmaceuticals, Bievres, France) administered daily by vaginal route starting on the evening of ET. Measurement of DNA Fragmentation by TUNEL Assay and Flow Cytometry From the ejaculate eventually used for IVF in each patient, a sperm aliquot (300–500 mL) was constituted to further DNA fragmentation assessment by the TUNEL assay (5– 7). Each of them contained at least 2 106 spermatozoa, which was diluted in 0.5 mL of phosphate-buffered saline (PBS) solution and then fixed by the addition of 1 mL 1% formaldehyde (Sigma Chemical Co., St. Louis, MO) for 60 minutes at room temperature. After 10-minute centrifugation, the pellet was resuspended in PBS and centrifuged again for 10 minutes. Afterwards, 10 mL sodium citrate 1% (MSD, Paris, France) with Triton X-100 (final concentration 0.1%; Supelco, Paris, France) was added to permeabilize cell membranes. After two more washes in PBS, the fixed permeabilized sperm was kept at 4 C in the dark until analysis.
Sperm Preparation, Fertilization, and Embryo Culture Sperm samples were collected by masturbation. Samples were analyzed for total sperm number, concentration, motility, and morphology. Sperm selection was done by migration on a PureSperm gradient (Nidacon; JCD, Lyon, France). Sperm was layered upon a 45%:90% PureSperm density gradient, processed by centrifuge at 600g for 15 minutes, and resuspended in 1 mL capacitating medium (FerticultFertipro; JCD).
For the assessment of DNA fragmentation, the final preparation was centrifuged for 10 minutes and the pellet diluted in 50 mL staining buffer containing 3mmol/L biotine 16dUTP and 10 U terminal deoxynucleotidyl transferase (TdT; Roche Diagnostics, Meylan, France) and incubated at 37 C for 45 minutes. Spermatozoa were then treated with streptavidin-fluorescein (Roche Diagnostics) following by incubation at room temperature in the dark for 40 minutes. Stained spermatozoa were washed twice in PBS and rediluted in 750 mL PBS containing 10 mg/mL propidium iodide (Sigma Chemical Co.). Negative control sperm cells were treated identically except for the omission of enzymatic solution. Positive control sperm cells were treated identically after exposition to DNAse I (Roche Diagnostics) for 30 minutes.
After density-gradient separation, a second evaluation of concentration, motility, and morphology was performed.
Sperm DNA fragmentation and propidium iodide labeling were analyzed with a flow cytometer (EPICS-XL6MCL;
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Beckman Coulter, Villepinte, France) equipped with a 15 mW argon-ion laser as the light source. The flow rate was set at approximately 500 spermatozoa/s, and 5,000 to 10,000 stained spermatozoa were analyzed in each sample. Light-scatter and fluorescence data were obtained at a fixed gain setting in logarithmic mode. The green fluorescence (FL1) signals were detected through a 530 nm band pass filter, and red fluorescence (PI) signals were collected through a 620 nm band pass filter. To distinguish spermatozoa from debris, a dot plot distribution of spermatozoa, based on linear forward scatter, which correlates with cell size, and side scatter, which correlates with cell density, was used. The data were processed on a computer using Lysis software. The percentage of DNA-fragmented sperm in each sample was determined by subtracting negative control green fluorescence histograms from TdT-positive ones. Definition of Sperm DNA Fragmentation Groups According to the percentage of DNA-fragmented sperm remaining inferior to or exceeding 35%, 2 study groups were determined: low fragmentation group (<35%, LF, n ¼ 65) and high fragmentation group (R35%, HF, n ¼ 52). The choice of 35% threshold was arbitrary and based on the fact that it corresponds to the rounded median value (50th percentile) of DNA fragmentation rate in the present population. Statistics The measure of central tendency used was the mean, and the measure of variability was the standard deviation. The 95% confidence intervals (CIs) were estimated when necessary.
When normality of data could not be ascertained, medians and minimum-maximum values were used. Differences of continuous variables in LF and HF groups were analyzed using the Student t test and those of proportions with the chi-squared test. The Mann-Whitney test was used for nonparametric data. When the P value was < .05, the difference was considered to be statistically significant. RESULTS Patients’ Characteristics and Ovarian Response to COH Patients’ characteristics in LF and HF groups are detailed in Table 1. As shown, female age, day 3 ovarian reserve assessment (serum FSH and inhibin B levels), rank of the current IVF-ET attempt, and indications for IVF-ET were similar in LF and HF groups. In addition, characteristics of the ovarian response to COH were similar in both groups. Sperm Characteristics Overall, median sperm characteristics met the usual criteria needed to perform conventional IVF-ET. Sperm concentration before PureSperm preparation was 20 (range 7–260) 106 spermatozoa/mL and after was 20 (range 7–276) 106 spermatozoa/mL; sperm motility before preparation was 40% (range 20%–60%) and after was 90% (range 60%–95%). As shown in Table 2, both groups (LF and HF) were comparable regarding semen parameters. Embryology Data and IVF-ET Outcome Embryology data and IVF-ET outcome in LF and HF groups are summarized in Table 3. Both groups were comparable regarding the total number of retrieved and inseminated oocytes, fertilization rates, and number of embryos obtained.
TABLE 1 Female population characteristics in the low (LF) and high (HF) fragmentation groups.
Age of women (yrs)a Basal FSH (mUI/mL)a Inhibin B (pg/mL)a Rank of IVF-ET attempt Tubal factor Grade I endometriosis Unexplained hMG requirement (UI) Day of hCG administration E2 level (pg/mL)b Number of follicles R16 mma,b Endometrial thickness (mm)b Number of ET
LF (n [ 65)
HF (n [ 52)
P value
33.1 [23–37] 5.9 [2.3–9.9] 77 [45–185] 2.02 1.5 37 (57%) 7 (11%) 21 (32%) 2477 801 11.9 0.2 3066 1160 8 [5–13] 9.8 0.2 64
33.3 [22–37] 6.2 [2.6–9.9] 74 [45–180] 1.71 1.03 32 (62%) 7 (13%) 13 (25%) 2621 819 11.9 0.2 2845 1130 7.5 [5–15] 9.7 0.3 51
NS NS NS NS NS NS NS NS NS NS NS NS —
Note: NS ¼ not significant. a Median [range]. b On the day of hCG administration. Frydman. Sperm DNA fragmentation and ART outcome. Fertil Steril 2008.
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In line with this, the prevalence of top embryos obtained, the number of transfered embryos, and the prevalence of topquality embryos transfered were similar in LF and HF groups. As also shown in Table 3, clinical pregnancy rates (gestational sac with positive heartbeat observed ultrasonographically at 7 weeks of amenorrhoea), ongoing pregnancy rates (>12 weeks of amenorrhoea) per OR, and implantation rates (total number of gestational sacs 100/total number of embryos transfered) were statistically higher in the LF group than in the HF group. Furthermore, miscarriage rates were statistically decreased in the LF compared with the HF group. DISCUSSION The present clinical investigation represents an attempt to test the hypothesis that healthy oocytes could be able to repair sperm DNA fragmentation (21, 22) and compensate for its reported adverse effects on IVF-ET results (10, 12, 24). Indeed, it is possible that a number of physiologic sperm functions are compromised by chromatin fragmentation at, at least, two levels. On the one hand, sperm DNA fragmentation may hinder some putative early paternal effects (6), such as adequate oocyte fertilization, embryo morphology, and implantation (5, 18, 25). On the other hand, late paternal functions may be altered by this condition as the sperm aptitude to generate embryos leading to viable pregnancies (6, 15, 17, 19). Yet, women endowed with adequate ovarian function, and presumably more likely to produce DNA-repairing oocytes, should display unaltered IVF-ET results despite increased sperm DNA fragmentation rates. In such a selected female population, we observed that oocyte fertilization rates and early embryo morphology remained unaltered. Yet, this lack of detrimental effect of high sperm DNA fragmentation
may not be exclusively attributed to the DNA-repairing aptitude, if any, of oocytes. Indeed, because the paternal genome is barely expressed before the third cycle of embryo cell division (26), it is unlikely that sperm DNA fragmentation could encumber oocyte fertilization or early embryo characteristics. Yet the issue of whether sperm DNA damage may affect later stages of blastomere division could not be addressed by the present study and remains to be investigated. A trend for reduced blastocyst development rate has been noticed in the presence of high sperm DNA fragmentation (exceeding 20%) in a preliminary report (25). Incidentally, those authors assessed the extent of DNA fragmentation by TUNEL assay in an unselected female population (25). In addition, even in women displaying healthy ovarian function, we observed significantly lower clinical pregnancy and embryo implantation rates in the HF group than in the LF group. A number of hypotheses may be considered to explain this result. First, it is conceivable that patient selection based merely on clinical criteria of adequate ovarian functioning was insufficient to thoroughly exclude oocytes with deficient DNA-repairing competence. Unfortunately, by design, adequate oocyte morphology could not be retained as an additional criterion for patient selection in the present investigation, because only cases of conventional IVF-ET were included. Yet, it is noteworthy that reliable criteria for identifying oocytes that are more prone to repair sperm DNA fragmentation are as yet not available. Second, it is possible that the oocyte, irrespective of its quality, is inherently unable to overcome unwanted effects of extensive sperm DNA damage on embryo viability. This pivotal issue deserves to be the matter of further basic and
TABLE 2 Sperm characteristics before and after preparation in the low (LF) and high (HF) fragmentation groups.
Before sperm preparationa Volume of semen Sperm concentration (106/mL) Vitality (%) Motility (% of global motility) Progressive motility (% of a þ b) Percentage of typical forms After sperm preparationa No. of progressive spermatozoa (106) Motility (% of global motility) Progressive motility (% of a þ b)
LF (n [ 65)
HF (n [ 52)
P value
3.5 [1.5–7.0] 17.1 [7–260] 70 [51–95] 40 [30–60] 30 [20–50] 46 [19–67]
3.5 [1.5–7.0] 17.8 [7–150] 70 [55–90] 40 [20–60] 30 [10–50] 40 [19–62]
NS NS NS NS NS NS
15 [1–112] 90 [70–95] 80 [60–90]
18 [2–84] 90 [60–95] 80 [50–90]
NS NS NS
Note: NS ¼ not significant. a Median [range]. Frydman. Sperm DNA fragmentation and ART outcome. Fertil Steril 2008.
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TABLE 3 Embryology data and IVF-ET outcome in the low (LF) and high (HF) fragmentation groups.
No. of oocytes retrieved No. of 2 pro-nuclei zygotes No. of 3 pro-nuclei zygotes Fertilization rate [95% CI] No. of embryos obtained Prevalence of top-quality embryos [95% CI] No. of transfered embryos Prevalence of top-quality embryos transfered [95% CI] Clinical pregnancy rate per OR [95% CI] Implantation rate [95% CI] Ongoing pregnancy rate per OR [95% CI] Miscarriage rate [95% CI] Live birth rate per OR [95% CI] Take-home babies
LF (n [ 65)
HF (n [ 52)
P value
11.7 0.6 7.0 0.5 1.0 0.1 69.9% [64.4–75.3] 7.9 0.5 62.2% [54.9–69.5] 2.1 0.06 93.9% [88.5–99.3] 62.5% [50.4–74.6] 42.4% [33.8–51.0] 57.8% [45.5–70.1] 10.0% [0.51–19.5] 56.2% [43.8–68.6] 46
11.3 0.6 7.2 0.5 0.9 0.1 71.7% [66.0–77.4] 7.4 0.5 62.5% [54.4–70.6] 2.1 0.06 91.2% [84.6–97.8] 37.5% [24.0–51] 24.5% [16.0–33.0] 23.5% [11.7–35.3] 36.8% [14.7–58.9] 23.5% [11.7–35.3] 16
NS NS NS NS NS NS NS NS .007 .0043 .0002 .01 .0004
Note: CI ¼ confidence interval; NS ¼ not significant; OR ¼ oocyte retrieval. Frydman. Sperm DNA fragmentation and ART outcome. Fertil Steril 2008.
clinical research. Further, the poorer IVF-ET outcome associated with extensive sperm DNA fragmentation may result from alteration of the embryo potential to reach the blastocyst stage (20, 25), to successfully implant into the uterus, or both. Yet the present results are not contributive to clarifying these issues which deserve additional investigation.
characteristics. Also, the development of clinical and biologic measures aiming at repairing sperm DNA pathologies and/or selecting healthy spermatozoa before IVF-ET should be considered to improve spontaneous or assisted fertility.
The present data also showed a more than threefold increase in miscarriage rates in women included in the HF group compared with the LF group, in agreement with data from other investigators (19). This strongly suggests an involvement of sperm DNA fragmentation in the development competence of the implanted embryo (late paternal effect). However, the mechanisms underlying this phenomenon remain unclear. It may be due to ‘‘abortive apoptosis,’’ a pathologic condition known to yield the persistence of high level of sperm DNA fragmentation in raw semen (27–29).
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