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Sperm DNA fragmentation levels in testicular sperm samples from azoospermic males as assessed by the sperm chromatin dispersion (SCD) test
Sperm DNA fragmentation levels in testicular sperm samples from azoospermic males as assessed by the sperm chromatin dispersion (SCD) test Marcos Meseguer, Ph.D.,a Rebeca Santiso, Ph.D.,b,c Nicolas Garrido, Ph.D.,a Manuel Gil-Salom, M.D.,a Jose Remohı, M.D.,a and Jose Luis Fernandez, M.D.b,c a
IVI, Universidad de Valencia, Valencia; b Seccion de Genetica y Unidad de Investigacion, Hospital ‘‘Teresa Herrera,’’ Complejo Hospitalario Universitario Juan Canalejo, A Coru~na; and c Centro Oncologico de Galicia, A Coru~na, Spain
Objective: To analyze sperm DNA fragmentation (SDF) in testicular sperm samples from patients with azoospermia either from spermatogenic failure or from duct obstruction. Several technologies can be applied in the evaluation of SDF, but given the ease and low costs, the sperm chromatin dispersion test (SCD) has emerged as a promising standard. Design: Prospective blind observational cohort study. Setting: University-affiliated private IVF setting. Patient(s): Azoospermic patients from couples undergoing intracytoplasmic sperm injection cycles. Intervention(s): Testicular sperm extraction (TESE). Main Outcome Measurement(s): We determined testicular SDF, and a basic comparison between nonobstructive (n ¼ 22) and obstructive azoospermia (n ¼ 40) was performed. We also correlated SDF with embryo quality and pregnancy outcome. Result(s): SDF in the testicular sperm of patients with nonobstructive azoospermia was significantly higher, 46.92% (SEM ¼ 4.47), than that of patients with obstructive azoospermia, 35.96% (SEM ¼ 2.63). A moderate relationship between embryo morphology and testicular SDF was detected. Logistic regression analysis of the effect of testicular SDF on pregnancy outcome revealed no significant effect (odds ratio ¼ 1.015). Conclusion(s): Ours is the first report of SDF analysis in testicular sperm by using SCD in azoospermia. This result suggests that spermatogenesis failure may result in a severe affectation of sperm DNA integrity. The degree of DNA fragmentation using the SCD test is not reflected in pregnancy chances, and the explanation could be that embryos have been selected. (Fertil Steril 2009;92:1638–45. 2009 by American Society for Reproductive Medicine.) Key Words: Testicular sperm, DNA fragmentation, spermatogenesis, embryo quality, SCD test
Several studies have shown that male fertility can be affected by sperm DNA damage (reviewed by Tarozzi et al.) (1). Our group has previously shown the direct implications of DNA fragmentation measured by the sperm chromatin dispersion (SCD) test in IVF outcome (2). Two interesting conclusions were derived from the results obtained in IVF cycles. First, the determination of sperm DNA fragmentation (SDF) could be of special interest for patients with low implantation rates and low embryo quality. The DNA damage could subsequently be a possible causal factor. Second, the observation of nucleolus asynchrony in a zygote from a sperm sample with a high DNA fragmentation could indicate the possibility Received June 2, 2008; revised and accepted August 20, 2008; published online November 11, 2008. M.M. has nothing to disclose. R.S. has nothing to disclose. N.G. has nothing to disclose. M.G.-S. has nothing to disclose. J.R. has nothing to disclose. J.L.F. has nothing to disclose. This work was presented in part at the 33rd meeting of the American Society of Andrology, Albuquerque, NM, April 2008. This work was supported by IMPIVA IMIDTF/2007/182 (Generalitat Valenciana) and Xunta de Galicia INCITE07PXI916201ES. Reprint requests: Marcos Meseguer, Ph.D., Instituto Valenciano de Infertilidad, Plaza de la Policıa Local, 3, Valencia 46015, Spain (FAX: 34-96-3050999; E-mail: [email protected]).
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of an upcoming low-quality blastocyst cohort. In addition, SDF appears to be related to the ability of sperm to fertilize the oocyte (3). If DNA damage detected in ejaculated spermatozoa essentially begins after sperm release from Sertoli cells, it can be hypothesized that the degree of damage increases after Sertoli cell release. If this is correct, sperm populations recovered directly from the testis could be likely less affected by this pathological process as compared with ejaculated sperm populations. In fact, Greco et al. showed that the incidence of DNA fragmentation was markedly lower in testicular spermatozoa compared with ejaculated spermatozoa, and in consequence these data suggest that intracytoplasmic sperm injection (ICSI) with testicular spermatozoa possibly offers a viable assisted reproduction treatment option for men with high levels of sperm DNA damage (4). Since this is probably the most simple hypothesis, the risk exists that sperm obtained before complete maturation may have chromosomal alterations. We do not have this information, but we know from the literature that this is quite typical in cases of oligozoospermia, azoospermia, teratospermia, and so on (5–7).
Fertility and Sterility Vol. 92, No. 5, November 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.08.106
In fact, we (8) performed simultaneous DNA fragmentation and chromosomal analyses on the same sperm cell and showed a higher frequency of DNA fragmentation in sperm cells containing sex chromosome aneuploidies that originated in both the first and second meiotic divisions. The observed increase may suggest that the occurrence of aneuploidy during sperm maturation leads to SDF as part of a genomic screening mechanism developed to genetically inactivate sperm with a defective genomic makeup. In other words, the complete sperm maturation through DNA damage could be acting as a biological selector of the sperm quality. Our aim in this study is to assess the levels of DNA fragmentation in testicular sperm by using the SCD test and to correlate them with spermatogenesis, sperm motility, and sperm morphology. Moreover, the developmental competence of testicular spermatozoa was determined by evaluating the fertilization rate, embryo quality, and implantation rate and predicting the reproductive outcome. MATERIALS AND METHODS Institutional Approval and Informed Consent The study was approved by the Institutional Review Board of the Instituto Valenciano de Infertilidad. Sperm samples for research were obtained after written consent from patients. Patients We analyzed testicular sperm samples from patients diagnosed with azoospermia who were referred to our unit to obtain testicular sperm by testicular sperm extraction (TESE) and subsequent cryopreservation during the period from January 2007 to April 2008, yielding a total number of 62 men whose clinical files were studied. Azoospermia was confirmed by at least three centrifuged semen pellet analyses. The mean age of our male patients was 35.9 years (SD ¼ 5.3 years). The mean age of our female population was 34.7 years (SD ¼ 4.3 years). The male indications of TESE were distributed as follows: vasectomy (n ¼ 19, 23.6%); deferent agenesis (n ¼ 12, 19.3%); anejaculation (n ¼ 5, 8.1%); epididimal inflammation (n ¼ 4, 6.5%); and nonobstructive azoospermia (n ¼ 22, 35.4%). The female etiology of infertility was as follows: normal (n ¼ 52, 83.9 %); and tubal obstruction (n ¼ 10, 16.1%). The E2 levels before hCG injection were 2349.2 pg/mL (SD ¼ 505.3 pg/mL). The mean characteristics of the testicular sperm samples were as follows (average of seminal characteristics including range in parentheses): number of sperm cells per field 2.67 (0.12–10) and number of motile sperm cells per field 0.19 (0–0.9). TESE and Testicular Sperm Cryopreservation TESE was performed as described elsewhere (9). Open testicular biopsies were carried out under 2% mepivacaine sperFertility and Sterility
matic cord block. After opening the scrotal skin and tunica vaginalis, one to three small incisions were made through the tunica albuginea in different regions of each testicle, and small pieces of extruding testicular tissue were excised. Two fragments (one per testis) were embedded within Bouin’s solution and were sent for histopathological examination. The remaining fragments were carried to the adjacent laboratory for sperm retrieval. Testicular tissue was placed in 2 mL of sperm preparation medium (Medicult; Jyllinge, Denmark) and minced mechanically with sterile slides. The presence of sperm cells was checked under an inverted microscope at 400 magnification. If the search result was positive for motile sperm, samples were immediately frozen by adding an equal volume of sperm freezing medium (Medicult; Jyllinge, Norway) containing glycerol and then homogenized and placed at room temperature for 10 minutes. These samples were frozen in small tablets using a dry ice surface for approximately 1 minute and then were plunged onto liquid nitrogen until future use (10). The number of sperm cells and of motile sperm cells per field (400 magnification) was recorded from each sample. For thawing, the pills were removed and transferred into 5-mL Falcon tubes and placed in an incubator at 37 C and 5% CO2. Then samples were washed again with sperm medium (Medicult) and centrifuged at 600 g for 5 minutes. The supernatant was discarded, and samples resuspended in 0.1–0.4 mL of sperm medium. Thereafter, motile spermatozoa were checked again for ICSI (11). A normal 46,XY karyotype was assessed in all patients suffering from nonobstructive azoospermia. In patients with congenital bilateral absence of vas deferens or idiopathic obstructions of the seminal tract, both partners were screened for cystic fibrosis transmembrane conductance regulator (CFTR) mutations. SDF Measurements Determination of DNA fragmentation was performed by the improved SCD test, using the Halosperm kit (INDAS Laboratories, Madrid, Spain). A sample aliquot was taken from each testicular sperm sample before banking. In brief, aliquots containing testicular sperm were taken in both fresh and frozen in liquid nitrogen. They were coded and sent to the Juan Canalejo Hospital, for processing and scoring, in a blind study. Gelled aliquots of low-melting-point agarose in Eppendorf tubes were provided in the kit, one to process each semen sample. Eppendorf tubes were placed in a water bath at 90–100 C for 5 minutes to fuse the agarose and then in a water bath at 37 C. After 5 minutes of incubation for temperature equilibration at 37 C, 60 mL of the thawed semen sample was added to the Eppendorf tube and mixed with the fused agarose. Twenty microliters of the semen-agarose mix were pipetted onto precoated slides, which were provided in the kit, and covered with a 22 22 mm coverslip. The slides were placed on a cold plate in the refrigerator (4 C) for 5 minutes to allow the agarose to 1639
produce a microgel with the sperm cells embedded within. The coverslips were gently removed, and the slides immediately immersed horizontally in an acid solution, which was previously prepared by mixing 80 mL of HCl from an Eppendorf tube in the kit with 10 mL of distilled water and incubated for 7 minutes. The slides were horizontally immersed in 10 mL of the lysing solution for 25 minutes. After washing 5 minutes in a tray with abundant distilled water, the slides were dehydrated in increasing concentrations of ethanol (70%, 90%, 100%) for 2 minutes each, air-dried, and stored at room temperature in opaque closed boxes. For bright-field microscopy, slides were horizontally covered with a mix of Wright’s staining solution (Merck, Darmstadt, Germany) and phosphate-buffered solution (1:1; Merck) for 5–10 minutes with continuous airflow. Then the slides were briefly washed in running water and allowed to dry. Strong staining is preferred to easily visualize the periphery of the dispersed DNA loop halos. The distilled water, ethanol, Wright staining solution (Merck 1.01383.0500), and phosphate-buffered solution (Merck 1.07294.1000) are not provided in the kit. However, these reagents are inexpensive and easy to obtain. A minimum of 500 spermatozoa per sample were scored under the 100 objective of the microscope (12). Five SCD patterns are established: [1] Sperm cells with large halos, that is, those whose halo width is similar or larger than the minor diameter of the core. [2] Sperm cells with medium-size halos, that is, those whose halo size is between large and very small halos. [3] Sperm cells with very small halos, that is, the halo width is similar to or smaller than 1/3 of the minor diameter of the core. [4] Sperm cells without a halo. [5] Sperm cells with halo degraded (weakly or irregularly stained). Sperm cells with very small halos, without halos, and without halo degradation contain fragmented DNA. Finally, nucleoids that do not correspond to sperm cells are separately scored. SDF was expressed as the percentage of sperm cells with fragmented DNA (13, 14). Ovarian Stimulation in the Assisted Reproduction Cycles For controlled ovarian hyperstimulation, only GnRH agonist protocols were used as described elsewhere (15). Briefly, patients started administration of 0.5 mg of leuprolide acetate (Procrin; Abbott, Madrid, Spain) in the midluteal phase of the previous cycle until negative vaginal ultrasound defined ovarian quiescence. The dose of GnRH agonist was then decreased to 0.25 mg until the day of hCG administration. The fixed starting dose of gonadotropins was 225 IU/day of FSH (Gonal-F; Serono, Madrid, Spain; or Puregon; Organon, Madrid, Spain) and/or human menopausal gonadotropin (Menopur; Ferring Pharmaceuticals, Madrid, Spain) for the first 2–5 days, when serum E2 was assessed and gonadotropin dose was adjusted according to a step-up or step-down protocol. When three or more follicles reached 18 mm in diameter, hCG (Ovitrelle, 250 mg; Serono) was administered, and oocyte retrieval was scheduled 36 hours later. Serum E2 and P 1640
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levels were measured in the morning of the hCG administration. Samples were tested with a microparticle enzyme immunoassay Axsym System (Abbott Cientifico S.A., Madrid, Spain). The serum E2 kit had a sensitivity of 28 pg/mL and intraobserver and interobserver variation coefficients of 6.6% and 7.7%, respectively. ART Procedures A total of 62 first ICSI cycles were studied. Recovered oocytes were inseminated using a micromanipulation technique (ICSI) as described elsewhere (16). Microinjected sperm were analyzed for sperm morphology by phase contrast in the inverted microscope and before microinjection at 400 magnification. When no motile sperm were found in the thawed testicular tissue, we used pentoxifylline (0.3 mM, 30 minutes, 37 C; Hemovas, Barcelona, Spain). A total of 459 oocytes were evaluated. Fertilization was assessed at 16–18 hours after ICSI by zygote pronuclear presence observation at 40 magnification under inverted microscope. A total of 331 oocytes were fertilized. Embryo cleavage was evaluated 24 hours after fertilization assessment, and embryos were transferred into the uterine cavity 72 (n ¼ 37) or 144 (n ¼ 25 blastocysts) hours after ICSI. An average of 1.89 embryos per patient were transferred (SD ¼ 0.983). Good-quality supernumerary embryos were frozen at day 3 (72 hours) or were cultured until day 6 (144 hours) for eventual future transfers. Clinical pregnancy was determined by observing a gestational sac with fetal heartbeat at 7 weeks of pregnancy. Embryo morphology (n ¼ 321) was evaluated on days 2 and 3 (48–72 hours), taking into account the number, symmetry of blastomeres, and percentage of fragmentation. Obviously, some of the embryo parameters could be calculated as an average of the embryo cohort per patient, and these include fertilization rate, embryo fragmentation, and average number of cells. Human blastocysts were scored on day 6 (144 hours) according to the expansion of the blastocoels cavity and the number and integrity of both the inner-cell mass and trophoectoderm cells, as described elsewhere (17). Statistical Analysis Parametric tests (t-test) were employed for comparisons between groups. Significance was defined as P<.05. Analysis of variance (ANOVA) was performed, and for multiple post hoc comparisons Bonferroni’s test was performed. Correlations between continuous embryo parameters and DNA fragmentation values were determined by regression analysis followed by ANOVA. P<.05 was considered statistically significant. We performed the statistical analysis following two main concepts: First (average concept), we calculated an average per patient from all zygote-embryo numerical data. These values were correlated with SDF. Specifically, we correlated basic sperm parameters, fertilization rate, embryo division, and Vol. 92, No. 5, November 2009
fragmentation and implantation rates by linear regression analysis. Second (individual concept), we used some embryo morphological patterns individually with respect to SDF values of the sperm samples. Specifically, we correlated embryo symmetry and the final destination of the embryos (nonviable, frozen embryo, and transferred embryo). Finally, we also performed a logistic regression analysis in which the effect of testicular SDF on pregnancy and abortion was quantified. A model was developed in which maternal age was included as a confounding factor. The significance was calculated by the omnibus test (likehood ratio), and the uncertainty that was explained by the model was evaluated by Negelkerke R2, which is a coefficient analogous to the R2 index of the linear regression analysis. The odds ratios of the effect of one unit of percent of testicular SDF on pregnancy and miscarriage outcome are expressed together with 95% confidence intervals (CIs), R2, and significance. Statistical analysis was performed using the Statistical Package for the Social Sciences, version 14 (SPSS Inc., Chicago) and MedCalc Software (Ghent, Belgium).
RESULTS Testicular Sperm Parameters and DNA Fragmentation SDF in testes presented an average of 37.70% of positive spermatozoa (95% CI, 33.21–42.19). After analyzing the results from testicular sperm and counting and correlating them with DNA fragmentation (percentage of fragmented cells), we did not found any significant correlation for sperm cells recovered per field (r ¼ 0.241, P¼.112) and motile sperm recovered per field (r ¼ 0.276, P¼.126). However, SDF analysis in testicular sperm with defective spermatogenesis (nonobstructive azoospermia) was significantly higher than that in patients with normal testicular sperm production (obstructive azoospermia; Fig. 1).
With respect to sperm morphology and its relationship with testicular SDF, we determined the microinjected sperm morphology (observed during ICSI procedure). We found a significant association, in which SDF was significantly higher in those patients in whom abnormal or borderline sperm cells were injected than in those with normal sperm cells (Fig. 1). We also compared the levels of SDF in those patients in whom pentoxifylline was used (n ¼ 15) in ICSI to induce sperm motility and detect live sperm. In those patients in whom pentoxifylline was used (defective sperm survival after thawing), SDF levels were considerably higher than in those patients in whom motile sperm were detectable after sperm thawing (Fig. 1).
Testicular SDF and Fertilization, Embryo Quality, and Implantation Rate Testicular SDF values were not correlated with fertilization rate per cycle (r ¼ 0.024 and P¼.864; Table 1). A total of 321 embryos were morphologically analyzed and correlated with testicular SDF levels from each patient. The number of blastomeres, percentage of embryo fragmentation, and symmetry of our embryo cohort were analyzed 48 and 72 hours after capacitation. On day 2 embryos (72 hours), an interesting association between embryo symmetry and SDF was discovered (Fig. 2). On day 3, the same observations were obtained. Regarding embryo fragmentation on days 2 and 3, no correlations were observed with respect to the percentage of testicular SDF (Table 1). Remarkable results were obtained by studying cell division 48 and 72 hours after fertilization, and on day 2 a negative linear association was found. This observation is quite significant because embryos with low numbers of cells on day 2 are associated with lower implantation rates (18). We classified embryos in long culture on day 6 of development as those that reached the blastocyst stage in comparison with those arrested during their development.
FIGURE 1 Graphic representation of the relationship between obstructive and nonobstructive azoospermia, sperm morphology and sperm recovery after thawing (pentoxifylline), and the percentage of testicular sperm with DNA fragmentation. Asterisks (*) denote a significant difference.
Meseguer. DNA fragmentation in azoospermia. Fertil Steril 2009.
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TABLE 1 Correlations among fertilization rate, implantation rate, average embryo fragmentation, and average number of cells 48 and 72 hours after fertilization with testicular SDF. R
P
Fertilization rate 0.024 .869 Blastomere number, 48 hours 0.163 .012a Blastomere number, 48 hours 0.266 .078 Embryo fragmentation, 72 hours 0.008 .914 Embryo fragmentation, 72 hours 0.015 .421 Implantation rate 0.067 .330 a
Denotes a significant linear relationship, P>.05.
Meseguer. DNA fragmentation in azoospermia. Fertil Steril 2009.
We compared testicular SDF levels in those patients who generated blastocysts with the rest of the cohort on day 6 (n ¼ 120). Approximately 47.93% of our embryo cohort reached the blastocyst stage (Fig. 3).
Additionally, we classified the embryos into three categories depending on their final destination, defining three categories: nonviable, frozen, and transferred embryos. With this codification we separated the embryos depending on their quality. Results of the grouped categories regarding testicular SDF show that increased DNA fragmentation is associated with a higher proportion of nonviable (bad-quality) embryos (Fig. 4). Finally, when implantation rates were also correlated with respect to sperm testicular SDF (Table 1), we did not find any significant correlation. Testicular SDF, Pregnancy, and Abortion The results of testicular SDF analysis were compared regarding pregnancy and abortion, and the sperm samples were divided into those able to initiate a pregnancy, those unable to initiate a pregnancy, and those who were pregnant but who suffered a clinical abortion. A total number of 23 clinical pregnancies were achieved in our study group, while 39 cycles were unsuccessful. However, there was no effect of testicular SDF in pregnancy probability as demonstrated by logistic regression
FIGURE 2 Percentage of cells with testicular SDF with respect to classification of embryos on day 3 depending on blastomere symmetry. We defined three types of embryos: type 1, embryo cell volume with equal distribution, which means similar size in even blastomere number embryos, that is, 4, 6, or 8, or one cell with bigger diameter in odd blastomere number embryos; type 3, embryos with one big cell, which is considered dominant and which represents at least 1/3 of the embryo volume; type 2, the rest of the embryos, which includes embryos with odd blastomere number and equal size and embryos with even or odd numbers but different blastomere sizes. Asterisks (*) denote a significant difference of the percentage of testicular SDF between the types considered (P< .05). A representative picture of each type of classified embryo has been included in the graphic.
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FIGURE 3 Classifications of blastocysts as those whose developed blastocoel cavity on day 6 (144 hours after fertilization) compare with the rest of the embryos that did not reach the blastocyst stage, which were considered to be arrested. A comparison of the percentage of cells with testicular SDF with respect to the blastocysts and the arrested embryos was performed. Asterisks (*) denote a significant difference in the percentage of testicular SDF between both types considered (P< .05). A representative picture of both types of classified embryos has been included in the graphic.
More significantly, these data also show that testicular SDF is associated with embryo quality after ICSI using testicular spermatozoa but not with ongoing clinical pregnancy, implantation, and fertilization rates. Our observations suggest that the impairment of embryo developmental competence caused by nuclear DNA damage becomes manifest intermediately in the preimplantation embryo development and continues during all the steps observed in the clinical embryology laboratory from 48 hours after fertilization. This sign of a sperm-derived developmental handicap is in agreement with previous observations showing that those embryos with less symmetry and a worse embryo score and that do not reach the blastocyst stage are associated with elevated SDF levels. In fact, we have described elsewhere that a paternal effect can be detected as early as the pronuclear-stage zygote (17, 19). However, this premature paternal effect is not associated with an increased testicular SDF (data not shown). These results are in agreement with our preceding studies showing that spermatozoa with DNA damage can still fertilize oocytes and give rise to morphologically ill-formed embryos. Nevertheless, these embryos will implant in the same proportions as well-formed embryos (3). Conversely, a negative correlation between SDF and fertilization rates has been also reported (20–22).
Meseguer. DNA fragmentation in azoospermia. Fertil Steril 2009.
analysis. We had a total of four abortions, and there was no association with testicular SDF (Table 2). DISCUSSION This is the first report of SDF analysis in testicular sperm by using SCD in azoospermic patients. The present data suggest that, in infertile men with azoospermia, the incidence of DNA damage in testicular sperm populations is much lower in the normal and active spermatogenic testis than in the testis with incomplete sperm production. This observation confirms the working hypothesis, in which part of the DNA damage observed results from alterations occurring at testicular level, that is, those associated with a defect in the earlier developmental phases. We still do not know why there is a difference between both azoopermia types, but SDF could be related to different principal causes in the testicle or in the ducts, and this means that an abnormal sperm genome or maybe apoptosis could be predominant in patients with defective spermatogenesis. On the other hand, the production of post-testicular SDF could have its origin in an excess of oxidative stress, and this may perhaps be predominant in the epididymal ducts of some patients. Obviously both situations (apoptosis and oxidative stress) could be acting in a synergistic fashion. Fertility and Sterility
It is interesting that the presumable bias in the correlations of SDF yield with pregnancy as a consequence of embryo selection for implantation. Usually, DNA-fragmented spermatozoa are used in ICSI treatments in human reproduction, and the use of fragmented sperm is still a matter of concern, mainly with respect to the long-term consequences on the development and behavior of generated offspring. Mouse experiments have also revealed that fragmented sperm cells reduce both the rate of preimplantation embryo development and the number of offspring. Experiments show a delay in the active demethylation of the male pronucleus and, moreover, affected gene transcription and methylation of some epigenetically regulated genes. Regarding the offspring, the females who were produced showed increased anxiety, lack of habituation pattern, deficit in short-term spatial memory, some large organs, and an increase in pathologies. In consequence, and keeping in mind these experiments, it is possible that, depending on the level of DNA fragmentation, oocytes may partially repair fragmented DNA, producing blastocysts able to implant and produce live offspring. The incomplete repair, however, may lead to long-term pathologies that in the case of humans are still not well documented (23). In addition to suggesting novel clues for a better understanding of male factor infertility in the severe cases and the processes involved, in this study, we show that the sperm morphology of testicular sperm cells (analyzed during ICSI) is clearly associated with SDF levels. The same observations can be related with sperm motility, as far as those samples that need pentoxifylline to induce sperm motility are associated with increased levels of SDF. 1643
FIGURE 4 Classification of the embryos into three categories depending on the final destination: nonviable, frozen embryo, and transferred embryo. Results from the compilation of these categories with testicular SDF show that increased SDF is associated with nonviable (bad-quality) embryos. Asterisks (*) denote a significant increase in the percentage of testicular SDF in those nonviable embryos produced compared with frozen and transferred embryos (good quality; P< .05).
Meseguer. DNA fragmentation in azoospermia. Fertil Steril 2009.
Diverse studies have suggested several threshold values for the percentage of damaged spermatozoa in the ejaculate above which sperm fertilization and developmental competence are compromised, apparently because of the use of different techniques for DNA damage visualization (24–26). We have been unable to detect this threshold, as we have published in our previous works (2, 3, 12, 13). Thus, from our point of view, the question of a clinically relevant threshold for SDF still remains open. Alternatively, the developmental impairment associated with SDF may be related to an earlier increased DNA oxidation, which could act by itself as an unfavorable factor in the embryo development course (19). Further studies are also needed to determine the mechanisms involved in the increased SDF observed in those patients with defective spermatogenesis. Research projects are undergoing to study the direct effect of the oxidative stress
TABLE 2 Logistic regression analysis of the effect of testicular SDF on pregnancy and abortion. Odds Ratio
95% CI
Pregnancy 1.009 0.973–1.045 Abortion 0.995 0.933–1.062
R2 Negelkerke
P
0.010 0.019
.807 .575
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on the DNA oxidation route produced in testicular sperm cells. This could be the primary source of DNA fragmentation, and maybe the administration of antioxidants to the patients could overcome this defect. Nonetheless, reports concerning the clinical usefulness of antioxidants in the treatment of male factor infertility are controversial (27). Even more, our results are against post-testicular damage on DNA, since we have observed high levels of SDF in testicular sperm and even higher levels in those coming from a nonobstructive azoospermia. In consequence, we do not completely agree with those investigators who postulate the utility of TESE in patients with high levels of SDF combined with severe oligozoospermia. In this situation, spermatogenesis is clearly affected, and subsequently, those sperm cells coming from testes would also have a high SDF. Another and important endpoint could be the development of in vitro selection techniques capable of enriching ejaculated sperm populations in healthy spermatozoa such as the electrophoresis system developed by Ainsworth et al. (28); the sperm suspensions generated by this system (even with testicular sperm) exhibited lower levels of DNA fragmentation. Our previous observations revealed that average values are comparable to levels of ejaculated sperm from infertile and cancer patients and higher than those from sperm donors (13). Even though this is a clinical study, the data obtained showed that SDF could operate as a predictor of testicular spermatogenesis. The degree of DNA fragmentation using an SCD test is related to the sperm production, but this is Vol. 92, No. 5, November 2009
not ultimately reflected in pregnancy chances, probably owing to the embryo selection bias.
REFERENCES 1. Tarozzi N, Bizzaro D, Flamigni C, Borini A. Clinical relevance of sperm DNA damage in assisted reproduction. Reprod Biomed Online 2007;14: 746–57. 2. Muriel L, Meseguer M, Fernandez JL, Alvarez J, Remohi J, Pellicer A, et al. Value of the sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination: a blind prospective study. Hum Reprod 2006;21:738–44. 3. 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 the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril 2006;85:371–83. 4. Greco E, Scarselli F, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, et al. Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Hum Reprod 2005;20:226–30. 5. Collodel G, Capitani S, Baccetti B, Pammolli A, Moretti E. Sperm aneuploidies and low progressive motility. Hum Reprod 2007;22:1893–8. 6. Faure AK, Aknin-Seifer I, Frerot G, Pelletier R, De Robertis C, Cans C, et al. Predictive factors for an increased risk of sperm aneuploidies in oligo-astheno-teratozoospermic males. Int J Androl 2007;30:153–62. 7. Fodor F, Kamory E, Csokay B, Kopa Z, Kiss A, Lantos I, et al. Rapid detection of sex chromosomal aneuploidies by QF-PCR: application in 200 men with severe oligozoospermia or azoospermia. Genet Test 2007;11: 139–45. 8. Muriel L, Goyanes V, Segrelles E, Gosalvez J, Alvarez JG, Fernandez JL. Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis. J Androl 2007;28:38–49. 9. Meseguer M, Garrido N, Remohi J, Pellicer A, Simon C, MartinezJabaloyas JM, et al. Testicular sperm extraction (TESE) and ICSI in patients with permanent azoospermia after chemotherapy. Hum Reprod 2003;18:1281–5. 10. Meseguer M, Garrido N, Martinez-Conejero JA, Simon C, Pellicer A, Remohi J. Role of cholesterol, calcium, and mitochondrial activity in the susceptibility for cryodamage after a cycle of freezing and thawing. Fertil Steril 2004;81:588–94. 11. Meseguer M, Garrido N, Remohi J, Pellicer A, Gil-Salom M. Testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) in hypogonadotropic hypogonadism with persistent azoospermia after hormonal therapy. J Assist Reprod Genet 2004;21:91–4. 12. Viloria T, Garrido N, Fernandez JL, Remohi J, Pellicer A, Meseguer M. Sperm selection by swim-up in terms of deoxyribonucleic acid fragmentation as measured by the sperm chromatin dispersion test is altered in heavy smokers. Fertil Steril 2007;88:523–5. 13. Meseguer M, Santiso R, Garrido N, Fernandez JL. The effect of cancer on sperm DNA fragmentation as measured by the sperm chromatin dispersion test. Fertil Steril 2008;90:225–7.
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14. Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl 2003;24:59–66. 15. Melo MA, Meseguer M, Garrido N, Bosch E, Pellicer A, Remohi J. The significance of premature luteinization in an oocyte-donation programme. Hum Reprod 2006;21:1503–7. 16. Garrido N, Meseguer M, Bellver J, Remohi J, Simon C, Pellicer A. Report of the results of a 2 year programme of sperm wash and ICSI treatment for human immunodeficiency virus and hepatitis C virus serodiscordant couples. Hum Reprod 2004;19:2581–6. 17. Meseguer M, de los Santos MJ, Simon C, Pellicer A, Remohi J, Garrido N. Effect of sperm glutathione peroxidases 1 and 4 on embryo asymmetry and blastocyst quality in oocyte donation cycles. Fertil Steril 2006;86:1376–85. 18. Gamiz P, Rubio C, de los Santos MJ, Mercader A, Simon C, Remohi J, et al. The effect of pronuclear morphology on early development and chromosomal abnormalities in cleavage-stage embryos. Hum Reprod 2003;18:2413–9. 19. Meseguer M, Martinez-Conejero JA, O’Connor JE, Pellicer A, Remohi J, Garrido N. The significance of sperm DNA oxidation in embryo development and reproductive outcome in an oocyte donation program: a new model to study a male infertility prognostic factor. Fertil Steril 2007. 20. 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. 21. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998;69:528–32. 22. 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. 23. Fernandez-Gonzalez R, Moreira PN, Perez-Crespo M, SanchezMartin M, Ramirez MA, Pericuesta E, et al. Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biol Reprod 2008;78: 761–72. 24. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, et al. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod 2007;22:174–9. 25. Benchaib M, Lornage J, Mazoyer C, Lejeune H, Salle B, Francois Guerin J. Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome. Fertil Steril 2007;87:93–100. 26. Sergerie M, Mieusset R, Daudin M, Thonneau P, Bujan L. Ten-year variation in semen parameters and sperm deoxyribonucleic acid integrity in a healthy fertile man. Fertil Steril 2006;86:1513.e11–8. 27. Agarwal A, Said TM. Carnitines and male infertility. Reprod Biomed Online 2004;8:376–84. 28. Ainsworth C, Nixon B, Jansen RP, Aitken RJ. First recorded pregnancy and normal birth after ICSI using electrophoretically isolated spermatozoa. Hum Reprod 2007;22:197–200.
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IVI, Universidad de Valencia, Valencia; b Seccion de Genetica y Unidad de Investigacion, Hospital ‘‘Teresa Herrera,’’ Complejo Hospitalario Universitario Juan Canalejo, A Coru~na; and c Centro Oncologico de Galicia, A Coru~na, Spain
Objective: To analyze sperm DNA fragmentation (SDF) in testicular sperm samples from patients with azoospermia either from spermatogenic failure or from duct obstruction. Several technologies can be applied in the evaluation of SDF, but given the ease and low costs, the sperm chromatin dispersion test (SCD) has emerged as a promising standard. Design: Prospective blind observational cohort study. Setting: University-affiliated private IVF setting. Patient(s): Azoospermic patients from couples undergoing intracytoplasmic sperm injection cycles. Intervention(s): Testicular sperm extraction (TESE). Main Outcome Measurement(s): We determined testicular SDF, and a basic comparison between nonobstructive (n ¼ 22) and obstructive azoospermia (n ¼ 40) was performed. We also correlated SDF with embryo quality and pregnancy outcome. Result(s): SDF in the testicular sperm of patients with nonobstructive azoospermia was significantly higher, 46.92% (SEM ¼ 4.47), than that of patients with obstructive azoospermia, 35.96% (SEM ¼ 2.63). A moderate relationship between embryo morphology and testicular SDF was detected. Logistic regression analysis of the effect of testicular SDF on pregnancy outcome revealed no significant effect (odds ratio ¼ 1.015). Conclusion(s): Ours is the first report of SDF analysis in testicular sperm by using SCD in azoospermia. This result suggests that spermatogenesis failure may result in a severe affectation of sperm DNA integrity. The degree of DNA fragmentation using the SCD test is not reflected in pregnancy chances, and the explanation could be that embryos have been selected. (Fertil Steril 2009;92:1638–45. 2009 by American Society for Reproductive Medicine.) Key Words: Testicular sperm, DNA fragmentation, spermatogenesis, embryo quality, SCD test
Several studies have shown that male fertility can be affected by sperm DNA damage (reviewed by Tarozzi et al.) (1). Our group has previously shown the direct implications of DNA fragmentation measured by the sperm chromatin dispersion (SCD) test in IVF outcome (2). Two interesting conclusions were derived from the results obtained in IVF cycles. First, the determination of sperm DNA fragmentation (SDF) could be of special interest for patients with low implantation rates and low embryo quality. The DNA damage could subsequently be a possible causal factor. Second, the observation of nucleolus asynchrony in a zygote from a sperm sample with a high DNA fragmentation could indicate the possibility Received June 2, 2008; revised and accepted August 20, 2008; published online November 11, 2008. M.M. has nothing to disclose. R.S. has nothing to disclose. N.G. has nothing to disclose. M.G.-S. has nothing to disclose. J.R. has nothing to disclose. J.L.F. has nothing to disclose. This work was presented in part at the 33rd meeting of the American Society of Andrology, Albuquerque, NM, April 2008. This work was supported by IMPIVA IMIDTF/2007/182 (Generalitat Valenciana) and Xunta de Galicia INCITE07PXI916201ES. Reprint requests: Marcos Meseguer, Ph.D., Instituto Valenciano de Infertilidad, Plaza de la Policıa Local, 3, Valencia 46015, Spain (FAX: 34-96-3050999; E-mail: [email protected]).
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of an upcoming low-quality blastocyst cohort. In addition, SDF appears to be related to the ability of sperm to fertilize the oocyte (3). If DNA damage detected in ejaculated spermatozoa essentially begins after sperm release from Sertoli cells, it can be hypothesized that the degree of damage increases after Sertoli cell release. If this is correct, sperm populations recovered directly from the testis could be likely less affected by this pathological process as compared with ejaculated sperm populations. In fact, Greco et al. showed that the incidence of DNA fragmentation was markedly lower in testicular spermatozoa compared with ejaculated spermatozoa, and in consequence these data suggest that intracytoplasmic sperm injection (ICSI) with testicular spermatozoa possibly offers a viable assisted reproduction treatment option for men with high levels of sperm DNA damage (4). Since this is probably the most simple hypothesis, the risk exists that sperm obtained before complete maturation may have chromosomal alterations. We do not have this information, but we know from the literature that this is quite typical in cases of oligozoospermia, azoospermia, teratospermia, and so on (5–7).
Fertility and Sterility Vol. 92, No. 5, November 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.08.106
In fact, we (8) performed simultaneous DNA fragmentation and chromosomal analyses on the same sperm cell and showed a higher frequency of DNA fragmentation in sperm cells containing sex chromosome aneuploidies that originated in both the first and second meiotic divisions. The observed increase may suggest that the occurrence of aneuploidy during sperm maturation leads to SDF as part of a genomic screening mechanism developed to genetically inactivate sperm with a defective genomic makeup. In other words, the complete sperm maturation through DNA damage could be acting as a biological selector of the sperm quality. Our aim in this study is to assess the levels of DNA fragmentation in testicular sperm by using the SCD test and to correlate them with spermatogenesis, sperm motility, and sperm morphology. Moreover, the developmental competence of testicular spermatozoa was determined by evaluating the fertilization rate, embryo quality, and implantation rate and predicting the reproductive outcome. MATERIALS AND METHODS Institutional Approval and Informed Consent The study was approved by the Institutional Review Board of the Instituto Valenciano de Infertilidad. Sperm samples for research were obtained after written consent from patients. Patients We analyzed testicular sperm samples from patients diagnosed with azoospermia who were referred to our unit to obtain testicular sperm by testicular sperm extraction (TESE) and subsequent cryopreservation during the period from January 2007 to April 2008, yielding a total number of 62 men whose clinical files were studied. Azoospermia was confirmed by at least three centrifuged semen pellet analyses. The mean age of our male patients was 35.9 years (SD ¼ 5.3 years). The mean age of our female population was 34.7 years (SD ¼ 4.3 years). The male indications of TESE were distributed as follows: vasectomy (n ¼ 19, 23.6%); deferent agenesis (n ¼ 12, 19.3%); anejaculation (n ¼ 5, 8.1%); epididimal inflammation (n ¼ 4, 6.5%); and nonobstructive azoospermia (n ¼ 22, 35.4%). The female etiology of infertility was as follows: normal (n ¼ 52, 83.9 %); and tubal obstruction (n ¼ 10, 16.1%). The E2 levels before hCG injection were 2349.2 pg/mL (SD ¼ 505.3 pg/mL). The mean characteristics of the testicular sperm samples were as follows (average of seminal characteristics including range in parentheses): number of sperm cells per field 2.67 (0.12–10) and number of motile sperm cells per field 0.19 (0–0.9). TESE and Testicular Sperm Cryopreservation TESE was performed as described elsewhere (9). Open testicular biopsies were carried out under 2% mepivacaine sperFertility and Sterility
matic cord block. After opening the scrotal skin and tunica vaginalis, one to three small incisions were made through the tunica albuginea in different regions of each testicle, and small pieces of extruding testicular tissue were excised. Two fragments (one per testis) were embedded within Bouin’s solution and were sent for histopathological examination. The remaining fragments were carried to the adjacent laboratory for sperm retrieval. Testicular tissue was placed in 2 mL of sperm preparation medium (Medicult; Jyllinge, Denmark) and minced mechanically with sterile slides. The presence of sperm cells was checked under an inverted microscope at 400 magnification. If the search result was positive for motile sperm, samples were immediately frozen by adding an equal volume of sperm freezing medium (Medicult; Jyllinge, Norway) containing glycerol and then homogenized and placed at room temperature for 10 minutes. These samples were frozen in small tablets using a dry ice surface for approximately 1 minute and then were plunged onto liquid nitrogen until future use (10). The number of sperm cells and of motile sperm cells per field (400 magnification) was recorded from each sample. For thawing, the pills were removed and transferred into 5-mL Falcon tubes and placed in an incubator at 37 C and 5% CO2. Then samples were washed again with sperm medium (Medicult) and centrifuged at 600 g for 5 minutes. The supernatant was discarded, and samples resuspended in 0.1–0.4 mL of sperm medium. Thereafter, motile spermatozoa were checked again for ICSI (11). A normal 46,XY karyotype was assessed in all patients suffering from nonobstructive azoospermia. In patients with congenital bilateral absence of vas deferens or idiopathic obstructions of the seminal tract, both partners were screened for cystic fibrosis transmembrane conductance regulator (CFTR) mutations. SDF Measurements Determination of DNA fragmentation was performed by the improved SCD test, using the Halosperm kit (INDAS Laboratories, Madrid, Spain). A sample aliquot was taken from each testicular sperm sample before banking. In brief, aliquots containing testicular sperm were taken in both fresh and frozen in liquid nitrogen. They were coded and sent to the Juan Canalejo Hospital, for processing and scoring, in a blind study. Gelled aliquots of low-melting-point agarose in Eppendorf tubes were provided in the kit, one to process each semen sample. Eppendorf tubes were placed in a water bath at 90–100 C for 5 minutes to fuse the agarose and then in a water bath at 37 C. After 5 minutes of incubation for temperature equilibration at 37 C, 60 mL of the thawed semen sample was added to the Eppendorf tube and mixed with the fused agarose. Twenty microliters of the semen-agarose mix were pipetted onto precoated slides, which were provided in the kit, and covered with a 22 22 mm coverslip. The slides were placed on a cold plate in the refrigerator (4 C) for 5 minutes to allow the agarose to 1639
produce a microgel with the sperm cells embedded within. The coverslips were gently removed, and the slides immediately immersed horizontally in an acid solution, which was previously prepared by mixing 80 mL of HCl from an Eppendorf tube in the kit with 10 mL of distilled water and incubated for 7 minutes. The slides were horizontally immersed in 10 mL of the lysing solution for 25 minutes. After washing 5 minutes in a tray with abundant distilled water, the slides were dehydrated in increasing concentrations of ethanol (70%, 90%, 100%) for 2 minutes each, air-dried, and stored at room temperature in opaque closed boxes. For bright-field microscopy, slides were horizontally covered with a mix of Wright’s staining solution (Merck, Darmstadt, Germany) and phosphate-buffered solution (1:1; Merck) for 5–10 minutes with continuous airflow. Then the slides were briefly washed in running water and allowed to dry. Strong staining is preferred to easily visualize the periphery of the dispersed DNA loop halos. The distilled water, ethanol, Wright staining solution (Merck 1.01383.0500), and phosphate-buffered solution (Merck 1.07294.1000) are not provided in the kit. However, these reagents are inexpensive and easy to obtain. A minimum of 500 spermatozoa per sample were scored under the 100 objective of the microscope (12). Five SCD patterns are established: [1] Sperm cells with large halos, that is, those whose halo width is similar or larger than the minor diameter of the core. [2] Sperm cells with medium-size halos, that is, those whose halo size is between large and very small halos. [3] Sperm cells with very small halos, that is, the halo width is similar to or smaller than 1/3 of the minor diameter of the core. [4] Sperm cells without a halo. [5] Sperm cells with halo degraded (weakly or irregularly stained). Sperm cells with very small halos, without halos, and without halo degradation contain fragmented DNA. Finally, nucleoids that do not correspond to sperm cells are separately scored. SDF was expressed as the percentage of sperm cells with fragmented DNA (13, 14). Ovarian Stimulation in the Assisted Reproduction Cycles For controlled ovarian hyperstimulation, only GnRH agonist protocols were used as described elsewhere (15). Briefly, patients started administration of 0.5 mg of leuprolide acetate (Procrin; Abbott, Madrid, Spain) in the midluteal phase of the previous cycle until negative vaginal ultrasound defined ovarian quiescence. The dose of GnRH agonist was then decreased to 0.25 mg until the day of hCG administration. The fixed starting dose of gonadotropins was 225 IU/day of FSH (Gonal-F; Serono, Madrid, Spain; or Puregon; Organon, Madrid, Spain) and/or human menopausal gonadotropin (Menopur; Ferring Pharmaceuticals, Madrid, Spain) for the first 2–5 days, when serum E2 was assessed and gonadotropin dose was adjusted according to a step-up or step-down protocol. When three or more follicles reached 18 mm in diameter, hCG (Ovitrelle, 250 mg; Serono) was administered, and oocyte retrieval was scheduled 36 hours later. Serum E2 and P 1640
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levels were measured in the morning of the hCG administration. Samples were tested with a microparticle enzyme immunoassay Axsym System (Abbott Cientifico S.A., Madrid, Spain). The serum E2 kit had a sensitivity of 28 pg/mL and intraobserver and interobserver variation coefficients of 6.6% and 7.7%, respectively. ART Procedures A total of 62 first ICSI cycles were studied. Recovered oocytes were inseminated using a micromanipulation technique (ICSI) as described elsewhere (16). Microinjected sperm were analyzed for sperm morphology by phase contrast in the inverted microscope and before microinjection at 400 magnification. When no motile sperm were found in the thawed testicular tissue, we used pentoxifylline (0.3 mM, 30 minutes, 37 C; Hemovas, Barcelona, Spain). A total of 459 oocytes were evaluated. Fertilization was assessed at 16–18 hours after ICSI by zygote pronuclear presence observation at 40 magnification under inverted microscope. A total of 331 oocytes were fertilized. Embryo cleavage was evaluated 24 hours after fertilization assessment, and embryos were transferred into the uterine cavity 72 (n ¼ 37) or 144 (n ¼ 25 blastocysts) hours after ICSI. An average of 1.89 embryos per patient were transferred (SD ¼ 0.983). Good-quality supernumerary embryos were frozen at day 3 (72 hours) or were cultured until day 6 (144 hours) for eventual future transfers. Clinical pregnancy was determined by observing a gestational sac with fetal heartbeat at 7 weeks of pregnancy. Embryo morphology (n ¼ 321) was evaluated on days 2 and 3 (48–72 hours), taking into account the number, symmetry of blastomeres, and percentage of fragmentation. Obviously, some of the embryo parameters could be calculated as an average of the embryo cohort per patient, and these include fertilization rate, embryo fragmentation, and average number of cells. Human blastocysts were scored on day 6 (144 hours) according to the expansion of the blastocoels cavity and the number and integrity of both the inner-cell mass and trophoectoderm cells, as described elsewhere (17). Statistical Analysis Parametric tests (t-test) were employed for comparisons between groups. Significance was defined as P<.05. Analysis of variance (ANOVA) was performed, and for multiple post hoc comparisons Bonferroni’s test was performed. Correlations between continuous embryo parameters and DNA fragmentation values were determined by regression analysis followed by ANOVA. P<.05 was considered statistically significant. We performed the statistical analysis following two main concepts: First (average concept), we calculated an average per patient from all zygote-embryo numerical data. These values were correlated with SDF. Specifically, we correlated basic sperm parameters, fertilization rate, embryo division, and Vol. 92, No. 5, November 2009
fragmentation and implantation rates by linear regression analysis. Second (individual concept), we used some embryo morphological patterns individually with respect to SDF values of the sperm samples. Specifically, we correlated embryo symmetry and the final destination of the embryos (nonviable, frozen embryo, and transferred embryo). Finally, we also performed a logistic regression analysis in which the effect of testicular SDF on pregnancy and abortion was quantified. A model was developed in which maternal age was included as a confounding factor. The significance was calculated by the omnibus test (likehood ratio), and the uncertainty that was explained by the model was evaluated by Negelkerke R2, which is a coefficient analogous to the R2 index of the linear regression analysis. The odds ratios of the effect of one unit of percent of testicular SDF on pregnancy and miscarriage outcome are expressed together with 95% confidence intervals (CIs), R2, and significance. Statistical analysis was performed using the Statistical Package for the Social Sciences, version 14 (SPSS Inc., Chicago) and MedCalc Software (Ghent, Belgium).
RESULTS Testicular Sperm Parameters and DNA Fragmentation SDF in testes presented an average of 37.70% of positive spermatozoa (95% CI, 33.21–42.19). After analyzing the results from testicular sperm and counting and correlating them with DNA fragmentation (percentage of fragmented cells), we did not found any significant correlation for sperm cells recovered per field (r ¼ 0.241, P¼.112) and motile sperm recovered per field (r ¼ 0.276, P¼.126). However, SDF analysis in testicular sperm with defective spermatogenesis (nonobstructive azoospermia) was significantly higher than that in patients with normal testicular sperm production (obstructive azoospermia; Fig. 1).
With respect to sperm morphology and its relationship with testicular SDF, we determined the microinjected sperm morphology (observed during ICSI procedure). We found a significant association, in which SDF was significantly higher in those patients in whom abnormal or borderline sperm cells were injected than in those with normal sperm cells (Fig. 1). We also compared the levels of SDF in those patients in whom pentoxifylline was used (n ¼ 15) in ICSI to induce sperm motility and detect live sperm. In those patients in whom pentoxifylline was used (defective sperm survival after thawing), SDF levels were considerably higher than in those patients in whom motile sperm were detectable after sperm thawing (Fig. 1).
Testicular SDF and Fertilization, Embryo Quality, and Implantation Rate Testicular SDF values were not correlated with fertilization rate per cycle (r ¼ 0.024 and P¼.864; Table 1). A total of 321 embryos were morphologically analyzed and correlated with testicular SDF levels from each patient. The number of blastomeres, percentage of embryo fragmentation, and symmetry of our embryo cohort were analyzed 48 and 72 hours after capacitation. On day 2 embryos (72 hours), an interesting association between embryo symmetry and SDF was discovered (Fig. 2). On day 3, the same observations were obtained. Regarding embryo fragmentation on days 2 and 3, no correlations were observed with respect to the percentage of testicular SDF (Table 1). Remarkable results were obtained by studying cell division 48 and 72 hours after fertilization, and on day 2 a negative linear association was found. This observation is quite significant because embryos with low numbers of cells on day 2 are associated with lower implantation rates (18). We classified embryos in long culture on day 6 of development as those that reached the blastocyst stage in comparison with those arrested during their development.
FIGURE 1 Graphic representation of the relationship between obstructive and nonobstructive azoospermia, sperm morphology and sperm recovery after thawing (pentoxifylline), and the percentage of testicular sperm with DNA fragmentation. Asterisks (*) denote a significant difference.
Meseguer. DNA fragmentation in azoospermia. Fertil Steril 2009.
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TABLE 1 Correlations among fertilization rate, implantation rate, average embryo fragmentation, and average number of cells 48 and 72 hours after fertilization with testicular SDF. R
P
Fertilization rate 0.024 .869 Blastomere number, 48 hours 0.163 .012a Blastomere number, 48 hours 0.266 .078 Embryo fragmentation, 72 hours 0.008 .914 Embryo fragmentation, 72 hours 0.015 .421 Implantation rate 0.067 .330 a
Denotes a significant linear relationship, P>.05.
Meseguer. DNA fragmentation in azoospermia. Fertil Steril 2009.
We compared testicular SDF levels in those patients who generated blastocysts with the rest of the cohort on day 6 (n ¼ 120). Approximately 47.93% of our embryo cohort reached the blastocyst stage (Fig. 3).
Additionally, we classified the embryos into three categories depending on their final destination, defining three categories: nonviable, frozen, and transferred embryos. With this codification we separated the embryos depending on their quality. Results of the grouped categories regarding testicular SDF show that increased DNA fragmentation is associated with a higher proportion of nonviable (bad-quality) embryos (Fig. 4). Finally, when implantation rates were also correlated with respect to sperm testicular SDF (Table 1), we did not find any significant correlation. Testicular SDF, Pregnancy, and Abortion The results of testicular SDF analysis were compared regarding pregnancy and abortion, and the sperm samples were divided into those able to initiate a pregnancy, those unable to initiate a pregnancy, and those who were pregnant but who suffered a clinical abortion. A total number of 23 clinical pregnancies were achieved in our study group, while 39 cycles were unsuccessful. However, there was no effect of testicular SDF in pregnancy probability as demonstrated by logistic regression
FIGURE 2 Percentage of cells with testicular SDF with respect to classification of embryos on day 3 depending on blastomere symmetry. We defined three types of embryos: type 1, embryo cell volume with equal distribution, which means similar size in even blastomere number embryos, that is, 4, 6, or 8, or one cell with bigger diameter in odd blastomere number embryos; type 3, embryos with one big cell, which is considered dominant and which represents at least 1/3 of the embryo volume; type 2, the rest of the embryos, which includes embryos with odd blastomere number and equal size and embryos with even or odd numbers but different blastomere sizes. Asterisks (*) denote a significant difference of the percentage of testicular SDF between the types considered (P< .05). A representative picture of each type of classified embryo has been included in the graphic.
Meseguer. DNA fragmentation in azoospermia. Fertil Steril 2009.
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FIGURE 3 Classifications of blastocysts as those whose developed blastocoel cavity on day 6 (144 hours after fertilization) compare with the rest of the embryos that did not reach the blastocyst stage, which were considered to be arrested. A comparison of the percentage of cells with testicular SDF with respect to the blastocysts and the arrested embryos was performed. Asterisks (*) denote a significant difference in the percentage of testicular SDF between both types considered (P< .05). A representative picture of both types of classified embryos has been included in the graphic.
More significantly, these data also show that testicular SDF is associated with embryo quality after ICSI using testicular spermatozoa but not with ongoing clinical pregnancy, implantation, and fertilization rates. Our observations suggest that the impairment of embryo developmental competence caused by nuclear DNA damage becomes manifest intermediately in the preimplantation embryo development and continues during all the steps observed in the clinical embryology laboratory from 48 hours after fertilization. This sign of a sperm-derived developmental handicap is in agreement with previous observations showing that those embryos with less symmetry and a worse embryo score and that do not reach the blastocyst stage are associated with elevated SDF levels. In fact, we have described elsewhere that a paternal effect can be detected as early as the pronuclear-stage zygote (17, 19). However, this premature paternal effect is not associated with an increased testicular SDF (data not shown). These results are in agreement with our preceding studies showing that spermatozoa with DNA damage can still fertilize oocytes and give rise to morphologically ill-formed embryos. Nevertheless, these embryos will implant in the same proportions as well-formed embryos (3). Conversely, a negative correlation between SDF and fertilization rates has been also reported (20–22).
Meseguer. DNA fragmentation in azoospermia. Fertil Steril 2009.
analysis. We had a total of four abortions, and there was no association with testicular SDF (Table 2). DISCUSSION This is the first report of SDF analysis in testicular sperm by using SCD in azoospermic patients. The present data suggest that, in infertile men with azoospermia, the incidence of DNA damage in testicular sperm populations is much lower in the normal and active spermatogenic testis than in the testis with incomplete sperm production. This observation confirms the working hypothesis, in which part of the DNA damage observed results from alterations occurring at testicular level, that is, those associated with a defect in the earlier developmental phases. We still do not know why there is a difference between both azoopermia types, but SDF could be related to different principal causes in the testicle or in the ducts, and this means that an abnormal sperm genome or maybe apoptosis could be predominant in patients with defective spermatogenesis. On the other hand, the production of post-testicular SDF could have its origin in an excess of oxidative stress, and this may perhaps be predominant in the epididymal ducts of some patients. Obviously both situations (apoptosis and oxidative stress) could be acting in a synergistic fashion. Fertility and Sterility
It is interesting that the presumable bias in the correlations of SDF yield with pregnancy as a consequence of embryo selection for implantation. Usually, DNA-fragmented spermatozoa are used in ICSI treatments in human reproduction, and the use of fragmented sperm is still a matter of concern, mainly with respect to the long-term consequences on the development and behavior of generated offspring. Mouse experiments have also revealed that fragmented sperm cells reduce both the rate of preimplantation embryo development and the number of offspring. Experiments show a delay in the active demethylation of the male pronucleus and, moreover, affected gene transcription and methylation of some epigenetically regulated genes. Regarding the offspring, the females who were produced showed increased anxiety, lack of habituation pattern, deficit in short-term spatial memory, some large organs, and an increase in pathologies. In consequence, and keeping in mind these experiments, it is possible that, depending on the level of DNA fragmentation, oocytes may partially repair fragmented DNA, producing blastocysts able to implant and produce live offspring. The incomplete repair, however, may lead to long-term pathologies that in the case of humans are still not well documented (23). In addition to suggesting novel clues for a better understanding of male factor infertility in the severe cases and the processes involved, in this study, we show that the sperm morphology of testicular sperm cells (analyzed during ICSI) is clearly associated with SDF levels. The same observations can be related with sperm motility, as far as those samples that need pentoxifylline to induce sperm motility are associated with increased levels of SDF. 1643
FIGURE 4 Classification of the embryos into three categories depending on the final destination: nonviable, frozen embryo, and transferred embryo. Results from the compilation of these categories with testicular SDF show that increased SDF is associated with nonviable (bad-quality) embryos. Asterisks (*) denote a significant increase in the percentage of testicular SDF in those nonviable embryos produced compared with frozen and transferred embryos (good quality; P< .05).
Meseguer. DNA fragmentation in azoospermia. Fertil Steril 2009.
Diverse studies have suggested several threshold values for the percentage of damaged spermatozoa in the ejaculate above which sperm fertilization and developmental competence are compromised, apparently because of the use of different techniques for DNA damage visualization (24–26). We have been unable to detect this threshold, as we have published in our previous works (2, 3, 12, 13). Thus, from our point of view, the question of a clinically relevant threshold for SDF still remains open. Alternatively, the developmental impairment associated with SDF may be related to an earlier increased DNA oxidation, which could act by itself as an unfavorable factor in the embryo development course (19). Further studies are also needed to determine the mechanisms involved in the increased SDF observed in those patients with defective spermatogenesis. Research projects are undergoing to study the direct effect of the oxidative stress
TABLE 2 Logistic regression analysis of the effect of testicular SDF on pregnancy and abortion. Odds Ratio
95% CI
Pregnancy 1.009 0.973–1.045 Abortion 0.995 0.933–1.062
R2 Negelkerke
P
0.010 0.019
.807 .575
Meseguer. DNA fragmentation in azoospermia. Fertil Steril 2009.
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DNA fragmentation in azoospermia
on the DNA oxidation route produced in testicular sperm cells. This could be the primary source of DNA fragmentation, and maybe the administration of antioxidants to the patients could overcome this defect. Nonetheless, reports concerning the clinical usefulness of antioxidants in the treatment of male factor infertility are controversial (27). Even more, our results are against post-testicular damage on DNA, since we have observed high levels of SDF in testicular sperm and even higher levels in those coming from a nonobstructive azoospermia. In consequence, we do not completely agree with those investigators who postulate the utility of TESE in patients with high levels of SDF combined with severe oligozoospermia. In this situation, spermatogenesis is clearly affected, and subsequently, those sperm cells coming from testes would also have a high SDF. Another and important endpoint could be the development of in vitro selection techniques capable of enriching ejaculated sperm populations in healthy spermatozoa such as the electrophoresis system developed by Ainsworth et al. (28); the sperm suspensions generated by this system (even with testicular sperm) exhibited lower levels of DNA fragmentation. Our previous observations revealed that average values are comparable to levels of ejaculated sperm from infertile and cancer patients and higher than those from sperm donors (13). Even though this is a clinical study, the data obtained showed that SDF could operate as a predictor of testicular spermatogenesis. The degree of DNA fragmentation using an SCD test is related to the sperm production, but this is Vol. 92, No. 5, November 2009
not ultimately reflected in pregnancy chances, probably owing to the embryo selection bias.
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