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

MALE FACTOR 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 Lourdes Muriel, Ph.D.,a Nicolás Garrido, Ph.D.,b José Luis Fernández, M.D.,a José Remohí, M.D.,b Antonio Pellicer, M.D.,b, c Maria José de los Santos, Ph.D.,b and Marcos Meseguer, Ph.D.b a

Sección de Genetica y Unidad de Investigación, Hospital “Teresa Herrera,” Complejo Hospitalario Universitario Juan Canalejo, A Corun˜a, Spain; b IVI Valencia, Universidad de Valencia, Valencia, Spain; and c Department of Obstetrics and Gynaecology, Hospital “Dr. Peset,” Valencia, Spain

Objective: To determine the prognostic value of sperm DNA fragmentation levels, as measured by the sperm chromatin dispersion (SCD) test, in predicting IVF and ICSI outcome. Design: Double-blind prospective study. Setting: University-affiliated private IVF setting. Patient(s): A total of 85 couples undergoing infertility treatment with IVF/ICSI. Intervention(s): Analysis of DNA fragmentation by the SCD test in 170 aliquots obtained from the ejaculate and from the processed semen used for assisted reproductive technologies (ART). Main Outcome Measure(s): Percentage of spermatozoa with fragmented DNA was statistically correlated with embryo quality and reproductive success. Result(s): Fertilization rate was inversely correlated with DNA fragmentation (r ⫽ ⫺0.245 P⫽.045). Higher DNA fragmentation rate gave an increased proportion of zygotes showing asynchrony between the nucleolar precursor bodies of zygote pronuclei (73.8% vs. 28.8% P⬍.001). In addition, the slower embryo development and worst morphology on day 6 was correlated with higher sperm DNA fragmentation (47.7% vs. 29.4% P⫽.044). We also observed a negative correlation between DNA fragmentation and the implantation rate (r ⫽ ⫺0.250 P⫽.042). However, SCD test values were not statistically different in cycles that resulted in a pregnancy compared with those that did not (33.2 vs. 28.2 and 32.4 vs. 34.7). Conclusion(s): This is the first report that describes a correlation between sperm DNA integrity, as measured by the SCD test, and fertilization rate, embryo quality, and implantation rate in IVF/ICSI. The degree of DNA fragmentation was inversely correlated with fertilization rate, synchrony of the nucleolar precursor bodies’ pattern in pronuclei, embryo ability to achieve blastocyst stage, and embryo morphological quality. Because SCD test values were correlated with embryo quality and blastocyst rate, the lack of correlation between sperm DNA fragmentation and pregnancy outcome in IVF might be due to embryo selection before transfer. The ability of the SCD test to predict the blastocyst rate after IVF/ICSI warrants further study. (Fertil Steril威 2006;85:371– 83. ©2006 by American Society for Reproductive Medicine.) Key Words: DNA fragmentation, sperm chromatin dispersion test, in vitro fertilization, embryo quality, sperm

A significant percentage of couples, even after several attempts of assisted reproduction, are still unable to conceive, although a thorough infertility evaluation has been performed and no apparent male or female factor has been identified. This could be related, at least in part, to some

Received April 12, 2005; revised and accepted July 27, 2005. Supported by Xunta de Galicia PGIDIT 04BTF916023PR grant. Reprint requests: Marcos Meseguer, Ph.D., IVI Valencia, Universidad de Valencia, IVF Laboratory; Plaza de la Policía Local 3, Valencia 46015, Spain. (FAX: 34 96 305 0999; E-mail: [email protected]).

0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.07.1327

sperm defects that are not being measured in the routine semen analysis. Such defects could be related to sperm DNA integrity. In fact, an increasing number of studies suggest that DNA fragmentation could be used as a marker of semen quality and a predictor of outcome in assisted reproductive technologies (ART) (1). It has been reported that about 10%– 20% of ejaculated spermatozoa have DNA fragmentation, and that apoptosis is more prevalent in oligozoospermic samples (2).

Fertility and Sterility姞 Vol. 85, No. 2, February 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.

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Dynamics of sperm DNA during spermatogenesis are subjected to a strict control (3), leading to mature nuclear spermatozoa that will be able to fertilize an oocyte. In contrast to the nucleus of somatic cells, the sperm nucleus contains one set of chromosomes (haploid) and the histones are replaced by transition proteins in early spermatids and by protamines in mature spermatids (4). In addition, DNA strands in sperm adopt a doughnut shape configuration that prevents DNA damage during sperm transport. Once the spermatozoon enters the egg, reducing factors from the egg’s cytoplasm reduce protamine disulphide bonds to sufhydryl groups (5), leading to chromatin decondensation and the formation of the male pronucleus. Some studies have suggested that failure of the oocyte to induce sperm nuclear decondensation could be a cause of disorders in the development of the male pronucleus (6). From these observations, it can be can concluded that chromatin configuration is stage-specific, and that alterations of this configuration could lead to defective spermatogenesis, subfertility, or infertility. A number of tests are currently available for the measurement of sperm DNA fragmentation (7). These include the TUNEL assay (8), the in situ nick translation, the comet assay (9), the chromomycin A3 test (10), the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) technique (11), and the sperm chromatin structure assay, (SCSA) (12). Independent of the method used, almost all of them have reported a negative correlation between sperm DNA fragmentation with fertilization rates and/or embryo development (13, 14). However, no effect, in terms of pregnancy rates, has been reported recently (14). The sperm chromatin dispersion (SCD) test is a novel assay based on an induced decondensation, which is directly linked with sperm DNA fragmentation. Intact human spermatozoa are immersed in an agarose matrix on a slide, treated with an acid solution to denature DNA that contains breaks, and then treated with a lysis buffer to remove sperm membranes and proteins giving rise to nucleoids with a central core and a peripheral halo of dispersed DNA loops. In human spermatozoa, sperm nuclei containing DNA fragmentation produce very small halos of DNA dispersion or there is an absence of these halos. These results were confirmed by sequential DBD-FISH, a procedure in which the restricted single-stranded DNA motifs generated from DNA breaks by the acid treatment can be detected and quantified (15). Thus, the presence or absence of DNA fragmentation can be determined merely by examining the halo size by SCD test, being a simple, highly reproducible and less expensive technique, with results highly correlated with those from other procedures like the SCSA (16). Recently, a thoroughly improved version has been developed as the Halosperm® kit, resulting in a much better chromatin quality as well as preservation of the tail. This allows a confident assessment of halo sizes under the conventional bright-field microscope, 372

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as well as a better discrimination of sperm cells from other cell types (16). The objective of this study was to prospectively evaluate the value of sperm DNA fragmentation testing, as measured by the SCD test, in predicting embryo quality and pregnancy outcome in IVF/ICSI. MATERIALS AND METHODS Institutional Approval and Informed Consent This project was approved by the institutional review board on the use of human subjects in research at the Instituto Valenciano de Infertilidad, and complies with the Spanish Law of Assisted Reproductive Technologies (35/1988). Sperm samples for research were obtained after written consent from patients. Patients Semen was obtained from male partners of couples who underwent IVF or ICSI for an infertility treatment. A total of 85 males provided 85 samples to be analyzed between April 2003 and September 2004. Patient ages ranged between 26 and 49; the median age was 36.8 ⫾ 3.1 years old in women and 37.6 ⫾ 2.7 in men. Semen Analysis Semen parameters were evaluated for every ejaculate by two independent observers. After the liquefaction of the semen at 37°C, 5% CO2 in air, for 10 minutes, the samples were examined for concentration and motility according to the WHO guidelines (WHO, 1999) on a Makler® chamber (Sefi Laboratories, Tel Aviv, Israel). Sperm morphology was studied following Tygerberg’s strict criteria. Spermatozoa were divided between normal and abnormal, and the latter were categorized depending on the type of defect (i.e., head defect, midpiece defect, and tail defect). Their respective percentages were then recorded (17). Results were only accepted when the differences between the two observers were less than 5%. Semen Preparation All samples were prepared by swim-up. Raw ejaculates were diluted 1:1 (vol:vol) with Sperm Medium (MediCult, Jyllinge, Denmark). Then, they were pelleted at 400 g for 10 minutes, and the supernatants were discarded. This was followed by the careful addition, without disturbing the pellet, of 0.5–1 mL of fresh medium, and the incubation for 45 minutes of the tubes with a 45° inclination. After this period, the upper 0.1– 0.5 mL was taken for the IVF or ICSI process. Two semen aliquots (from 10 –100 ␮L) were taken from each semen sample before and after sperm capacitation to be processed for the SCD assay. Ovarian Stimulation in the Assisted Reproduction Cycles For ovarian stimulation, both GnRH agonist and antagonist protocols were used. For GnRH agonist, long protocol was

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employed as previously described (18). Patients started administration of 0.1 mg of leuprolide acetate (Procrin; Abbott S.A., Valencia, Spain) or triptorelin (Decapeptyl; Ipsen Pharma, Valencia, Spain) in the midluteal phase of the previous cycle, until negative vaginal ultrasound defined ovarian quiescence. The dose of GnRH analogue was then decreased to 0.05 mg until the day of hCG administration. The GnRH antagonists were used following the low-dose daily protocol (18): Starting on stimulation day 6, 0.25 mg of the GnRH antagonist Cetrotide (Cetrorelix; Serono S.A., Valencia, Spain) was administered on a daily basis until the day of hCG administration. Recombinant FSH (Gonal-F; Serono S.A., Valencia, Spain; or Puregon; Organon Española, Valencia, Spain) and hMG (Lepori; Farma Laboratories, Valencia, Spain; or Menopur; Ferring, Valencia, Spain) were used for ovarian stimulation. Initial doses were determined according to patients’ age and basal serum FSH and estradiol (E2) levels. On stimulation day 3, serum E2 level was assessed and gonadotrophin doses adjusted according to a step-up or step-down protocol. The hCG (Profasi 10,000 UI; Serono S.A., Valencia, Spain) was administered when three or more follicles reached 18 mm in diameter and oocyte retrieval was scheduled 36 hours later. Oocyte Insemination Techniques Recovered oocytes were inseminated using either conventional insemination procedures, such as IVF, or micromanipulation techniques, such as ICSI, as previously described (19). Morphologically normal spermatozoa were sought in the sperm droplet, and then immobilized and aspirated, tail first, into the tip microinjection pipette. A metaphase II oocyte was held on the holding pipette, and the injection pipette was pushed through the zona pellucida injecting a single spermatozoon. Injected or inseminated oocytes were incubated in 20 ␮L drops. Fertilization was assessed after 18 hours, and embryo cleavage was assessed 24 hours thereafter. Embryos were transferred into the uterine cavity 48 –72 or 120 –144 hours after ICSI. Supernumerary embryos were frozen for eventual future transfers. Clinical pregnancy was determined by observing a gestational sac with fetal heartbeat at 7 weeks of pregnancy. Oocyte Fertilization Assessment Fertilization was assessed at 16 –18 hours post-IVF/ICSI. A total of 1,013 oocytes were evaluated. Zygote pronuclear morphology was observed at ⫻40 magnification under inverted microscope as described by Gamiz et al. (20). Two groups were formed on the basis of pronuclei size: those of equal or very similar size (group A) and those of different sizes (group B). In each group, zygotes were subdivided into four categories according to the number, distribution, and synchrony of nucleoli precursor bodies (NPB) (Fig. 1): subFertility and Sterility姞

group I, pronuclei with 3 ⫾ 4 polarized NPB; subgroup II, 7 ⫾ 3 synchronic polarized NPB or 7 ⫾ 3 NPB distributed randomly throughout the pronucleus; subgroup III, 1 or 2 NPB in one of the pronucleus; subgroup IV, morphologies other than those of groups I, II, or III (asynchronic NPB polarization, alignment of more than 10 NPB at the point of contact of the two pronuclei, a difference in more than three NPB between pronuclei, and a random distribution of ⬍4 NPB in both pronuclei). The presence of a cytoplasmic halo was also evaluated, classifying zygotes as halo-positive when they showed a perinuclear condensation of the cytoplasm and halo-negative when this polarization of the cytoplasm did not exist. Embryo Morphology-Quality Assessment Embryo morphology was evaluated on days 2 and 3 taking into account the number, symmetry, and granularity of blastomeres, type and percentage of fragmentation, presence of multinucleated blastomeres, and compaction degree. Human blastocysts were scored on day 5 and 6 (120 and 144 hours) according to the expansion of the blastocoele cavity and the number and integrity of both the inner cell mass (ICM) and trophoectoderm (T) cells, as described previously (21). We defined three groups: group a, complete T and high cell number compact ICM; group b, incomplete T and several grouped cells; group C, few cells in T or ICM. We defined optimal or good quality blastocysts as those where ICM and T are included in the a or b classification group. This means that T and ICM are complete and with compact and high enough cells, respectively. Obviously, some of the embryo parameters could be calculated similar to an average of the embryo cohort per patient, including: fertilization rate, embryo fragmentation, and average number of cells on day 3. SCD Test The improved SCD test has been developed using the Halosperm® kit (INDAS Laboratories, Madrid, Spain) (16). Aliquots containing 3–5 million sperm were taken in both fresh and prepared samples and frozen in liquid nitrogen. They were coded and sent to the Juan Canalejo Hospital for processing and scoring, in a double-blind study. Gelled aliquots of low-melting-point agarose in Eppendorf tubes were provided in the kit, each one to process a 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 ␮L of the thawed semen sample were added to the Eppendorf tube and mixed with the fused agarose; 20 ␮L of the semen-agarose mix were pipetted onto precoated slides, 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 produce a microgel with the sperm cells embedded within. 373

FIGURE 1 Pronuclear test according to nucleolar precursor bodies number and distribution. Description is included in each category.

Muriel. Sperm DNA fragmentation and IVF/ICSI outcome. Fertil Steril 2006.

The coverslips were gently removed and the slides immediately immersed horizontally in an acid solution, which had been previously prepared by mixing 80 ␮L of HCl from an Eppendorf tube included 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 buffer solution (Merck) (1:1) for 5–10 minutes with continuous airflow. Then the slides were briefly washed in running water for 10 seconds 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, Darmstadt, Germany), and phosphate buffer solution (Merck, Darmstadt, Germany) 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. 374

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Five SCD patterns were established (16): [1] Sperm cells with large halos: halo width is similar or higher than the minor diameter of the core [2] Sperm cells with medium-size halos: halo size is between those with high and with very small halo [3] Sperm cells with very small-size halo: halo width is similar or smaller than 1/3 of the minor diameter of the core [4] Sperm cells without a halo [5] Sperm cells without a halo-degraded: similar to item 4 but 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. A control microgel, containing a same sperm sample was enclosed in each slide. Statistical Analysis Parametric tests (t-test) were employed for comparisons between groups when the data followed a normal distribution. Significance was defined as P⬍.05. Analysis of variance (ANOVA) was performed and for multiple post hoc comparisons the Scheffe, DMS and Bonferroni’s test were performed. Correlation between continuous embryo parameters

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TABLE 1 Correlations between fertilization rate, average embryo fragmentation, and average number of cells on day 3 per patient with sperm DNA fragmentation on fresh and capacitated samples. DNA fragmentation average per patient Fertilization rate Average no. of blastomeres Embryo fragmentation Implantation rate

Fresh

Capacitated

R

P value

R

P value

⫺0.040 ⫺0.063 0.116 0.123

0.379 0.312 0.182 0.379

⫺0.241 ⫺0.041 0.088 ⫺0.250

0.045 0.396 0.282 0.042

Muriel. Sperm DNA fragmentation and IVF/ICSI outcome. Fertil Steril 2006.

and DNA fragmentation were performed by regression analysis followed by ANOVA. Significance was defined as P⬍.05. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL) and MedCalc Software (Ghent, Belgium). RESULTS The mean age of women included in the study was 34.8 ⫾ 3.3 years. The female etiology of infertility was unexplained for 16 couples (18.8%), artificial insemination failure for 18 couples (21.1%), tubal factor for 12 couples (14.1%), low response for 4 couples (4.7%), anovulation for 3 couples (3.5%), endometriosis for 7 couples (8.2%), ART failure for 6 couples (7.0), age for 6 couples (7.0%), recurrent abortion for 4 couples (4.7%) and other indications for 9 couples (10.5%). The E2 levels before hCG injection were 2,039.6 ⫾ 203.2 pg/mL.

ization rate (r ⫽ ⫺0.241, P⫽.045. Therefore, high DNA fragmentation yielded lower fertilization rate after IVF/ICSI (Table 1) (Fig. 2). In those zygotes that developed to the pronuclear stage, triploid zygotes were mainly found from samples with lower fragmented DNA levels, in both raw semen and processed sperm samples (Fig. 3A). The relationship between sperm DNA fragmentation and zygote score was also analyzed. A total number of 769 fertilized oocytes were then studied for pronuclei score, pronuclei symmetry, and presence of cytoplasmic halo. The

FIGURE 2 Linear regression analysis of the relationship between DNA fragmentation (x-axis) in capacitated sperm cells and fertilization rate (y-axis). Panel represents a scattergram and the regression line.

The mean characteristics (i.e., the average of seminal characteristics before and after semen processing) of the semen samples were as follows: fresh; volume: 3.2 mL (range, 0.4 –10.5), sperm concentration: 28.1 million (range, 0.18 –160), progressive motility: 14.5% (range, 4 – 80), morphology: 4.4% of normal forms (range, 2–30); capacitated; volume: 0.13 mL (range, 0.1–1.5), sperm concentration: 8.4 million/mL (range, 0.9 – 68), progressive motility: 13.7% (range, 7–96), morphology: 2.5% (range, 2–9). The mean characteristics of our embryo cohort were as follows (mean ⫾ SEM): oocytes retrieved per patient 12.84 ⫾ 0,78; fertilization rate 74.51 ⫾ 2.55; number of blastomeres on day three 6.61 ⫾ 0.19; percentage of embryo fragmentation on day three 16.60 ⫾ 1.25; number of embryos transferred per patient 1.80 ⫾ 0.10, implantation rate 29.41 ⫾ 5.03 and finally mean of frozen embryos per patient were 1.43 ⫾ 0.09. Sperm DNA Fragmentation and Fertilization Sperm DNA fragmentation of both fresh and capacitated sperm samples showed a significant negative correlation with fertilFertility and Sterility姞

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FIGURE 3 Graphic representation of the relationship between fresh and capacitated DNA fragmentation and zygote patterns. (A) Correlation of the number of pronucleus of each zygote with DNA fragmentation (PN ⫽ pronucleus). (B) DNA fragmentation is represented according with the PN patterns. (C) Regarding pronuclei size, we compare DNA fragmentation in those of equal or very similar size and those of different sizes. Asterisks (ⴱ) denote a significant increase of DNA fragmentation on the type IV pattern compared with the other three patterns (P⬍.05).

Muriel. Sperm DNA fragmentation and IVF/ICSI outcome. Fertil Steril 2006.

relationship between DNA fragmentation in fresh and capacitated samples and zygote patterns is detailed in Figure 3B and 3C. The percentage of sperm cells with fragmented DNA was significantly higher in group IV compared with 376

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the other three pronuclear patterns (Fig. 3B). The correlation with the presence or not of a perinuclear cytoplasmic halo was analyzed as an independent parameter with the identification of 487 halos positive and 41 halo negative. Slight

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FIGURE 3 CONTINUED

Muriel. Sperm DNA fragmentation and IVF/ICSI outcome. Fertil Steril 2006.

differences were found between them, but these differences were not statistically different in both fresh and capacitated sperm (data not shown). Regarding pronuclei size, we observed that asymmetric nuclei were more frequent when using capacitated sperm with higher DNA fragmentation degrees (Fig. 3C). Sperm DNA Fragmentation, Embryo Quality, and Implantation Rate Number of blastomeres, percentage and type of embryo fragmentation, symmetry, and multinucleation of our embryo cohort, were analyzed in day two (data not shown) and three, no effect was seen (Table 1). Although a slight positive relation between the frequency of sperm DNA fragmentation and embryo fragmentation was found, no effect was seen in terms of number of blastomeres, symmetry, or multinucleation. Otherwise, embryos coming from semen samples with higher DNA fragmentation level had significantly lower ability to develop fully expanded blastocysts on day 6 of development (Fig. 4B). Nevertheless, we did not observe any association between DNA fragmentation and the number and integrity of both inner cell mass and trophoectoderm cells of blastocysts (Table 2). We classified as optimal or good quality blastocysts as those where the ICM and T are included in a or b classification group, meaning that T and ICM are complete, and with compacted and high enough cells, respectively. We compared DNA fragmentation levels in blastocysts classified as optimal with the rest of the cohort on day 6. Approximately 17.5% of our blastocysts cohort was classiFertility and Sterility姞

fied as optimal. An association between embryo quality and sperm DNA fragmentation was found, although it did not reach statistical significance (Fig. 5). Finally, implantation rate was also correlated with sperm DNA fragmentation (Table 1). A significant negative correlation was found between the implantation rate in processed sperm (r ⫽ ⫺0.250, P⫽.042) and the DNA fragmentation level. Thus, an increase in sperm DNA fragmentation is associated with a reduction in implantation rate. Sperm DNA Fragmentation and Pregnancy The results of the sperm DNA fragmentation analysis were compared regarding pregnancy, dividing the sperm samples in those able to initiate a pregnancy, and those unable to do that. As reflected in Figure 6, samples were again divided into raw semen and sperm samples after swim-up. A total 34 pregnancies was achieved in our study, whereas 50 cycles were unsuccessful. The ROC curve analysis was performed to determine the predictive value of DNA fragmentation to achieve pregnancy. Results are presented in Table 3, indicating that none of the data had good area under the curve (AUC) values for pregnancy prediction in IVF cycles.

DISCUSSION It is widely accepted that sperm DNA fragmentation can be caused by defects in chromatin remodeling during spermiogenesis (8, 10, 22) and apoptosis during meiosis I. In fact, 377

FIGURE 4 Graphic representation of the scoring of the embryo on day 5 and day 6 and the association with DNA fragmentation. Degree of development on day 5 (A) and day 6 (B). Asterisks (ⴱ) denote a significant decrease of fresh DNA fragmentation on the expanded blastocyst pattern compared with blocked or morula stage embryos (P⬍.05) as well as a decrease in capacitated DNA fragmentation comparing a blastocyst, with its initial cavity, with an expanded blastocyst. CM ⫽ compact morula; EaB ⫽ early blastocyst; CB ⫽ blastocyst with initial cavity; EB ⫽ expanded blastocyst; HB ⫽ hatching blastocyst.

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TABLE 2 Day 6 human embryo scores, according to the number and integrity of both the ICM and trophoectoderm cells. ICM DNA fragmentation Day 5 Fresh Capacitated Day 6 Fresh Capacitated

Trophoectoderm

a

b

c

a

b

c

36.1 ⫾ 4.3 33.9 ⫾ 4.2

33.2 ⫾ 3.3 31.7 ⫾ 8.2

30.0 ⫾ 5.3 30.0 ⫾ 5.3

34.0 ⫾ 3.8 27.6 ⫾ 3.5

31.7 ⫾ 2.5 31.7 ⫾ 2.5

27.8 ⫾ 5.6 27.8 ⫾ 2.5

34.2 ⫾ 3.6 18.4 ⫾ 3.9

26.1 ⫾ 3.1 50.9 ⫾ 6.9

33.3 ⫾ 3.7 28.3 ⫾ 7.1

32.7 ⫾ 3.6 29.2 ⫾ 4.9

30.4 ⫾ 2.9 45.9 ⫾ 7.4

30.1 ⫾ 7.0 35.4 ⫾ 5.6

Note: The relationship between DNA fragmentation and the number and integrity of both ICM and trophoectoderm cells was studied. ICM ⫽ inner cell mass. Muriel. Sperm DNA fragmentation and IVF/ICSI outcome. Fertil Steril 2006.

Fas receptors have been found in ejaculated sperm of oligozoospermic samples (23). Moreover, activated caspases 1, 3, and 8 have been identified in the postacrosomal region of the sperm head and in the midpiece of immature sperm cells (caspase 9) (24, 25). The oxidative stress may also be a cause of sperm DNA fragmentation (26). It has been demonstrated that the immune seminal cells, immature germ cells, and mature sperm, contribute to the production of reactive oxygen species

(ROS) that are able to cause DNA damage (3). Several studies have reported that redox balance is deregulated in the ejaculates from infertile males, with glutathion peroxidase 4 being one of the main enzymes involved in this issue (27). Several studies have demonstrated that sperm DNA integrity correlates with pregnancy outcome in vitro fertilization (12, 28 –30). Therefore, sperm DNA fragmentation analysis should be included in the evaluation of the infertile male (7).

FIGURE 5 Good quality blastocysts were classified as those where the trophoectoderm and inner cell mass (ICM) were complete and with compact and high enough cells, respectively. A comparison of DNA fragmentation of these blastocysts with the rest of the cohort on day 6 was performed.

Muriel. Sperm DNA fragmentation and IVF/ICSI outcome. Fertil Steril 2006.

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FIGURE 6 Graphic representation of DNA fragmentation in raw and capacitated sperm samples used in the cycles. Samples were divided into two groups: pregnancy or not in the proper cycle. Data were combined and expressed as the mean ⫾ SEM. No statistical differences were found between both groups as compared using the student’s t-test.

Muriel. Sperm DNA fragmentation and IVF/ICSI outcome. Fertil Steril 2006.

The SCD test is a simple, fast, accurate, and reproducible method for the analysis of sperm DNA fragmentation. SCD test results are highly correlated with those obtained by DBD-FISH and the SCSA test, currently considered the gold standard in DNA fragmentation analysis (16). Our study is the first to report that sperm DNA fragmentation values, as measured by the SCD test, are negatively correlated with fertilization rate and embryo quality in IVF/ICSI. These results are particularly strengthened because:

[2] The analysis was performed in a blind fashion. [3] A regression analysis model was used. [4] A large number of samples were included in the study (n ⫽ 84). The clinical protocols, the ovarian response, the number of embryos transferred, and other IVF clinical data have been reported. These values can be considered as normal findings demonstrating the absence of female factors on the results.

[1] DNA fragmentation was determined in the actual sperm sample used for ART.

There was a negative correlation between fertilization rate and DNA fragmentation level, suggesting that sperm con-

TABLE 3 Diagnostic accuracy of DNA fragmentation in fresh and capacitated semen with respect to forecasting successful and unsuccessful pregnancy. Sperm DNA fragmentation Fresh Capacitated

AUCROC

Significance

Threshold

Sensitivity (%)

Specificity (%)

PPV

NPV

0.597 0.527

0.164 0.724

18.8 32.8

39.0 55.9

81.7 54.9

55.2 45.2

69.9 65.1

Note: AUCROC ⫽ area under the curve in a receiver operating characteristic analysis; PPV ⫽ positive predictive value; NPV ⫽ negative predictive value. Muriel. Sperm DNA fragmentation and IVF/ICSI outcome. Fertil Steril 2006.

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taining fragmented DNA may have difficulties to develop the pronuclear stage. Regarding embryo morphology, a thorough study was performed to analyze all the developmental stages. Previous publications stated that zygotes with three to seven nucleolar polar bodies in each pronucleus present the best prognosis (20). Interestingly, this good prognosis appears associated with lower DNA fragmentation level. The same observation was obtained when similar pronuclei size were observed. Moreover, an association between low DNA fragmentation and tendency to develop triploid zygotes was found. It is possible to speculate that polyspermia is favored when DNA fragmentation is lower because fertilization is decreased when sperm DNA is fragmented. Even so, this phenomenon is observed in IVF or ICSI unevenly and other cellular events should be responsible of this effect. We have not been able to correlate embryo morphology, development velocity or embryo fragmentation on day three with sperm DNA fragmentation. Nevertheless, the achievement of a later phase (i.e., blastocyst stage), as well as its morphological quality, was associated with lower sperm DNA fragmentation level. This occurred despite the fact that correlation with ICM and trophoblast cells morphology was not reflected in the analysis. The results indicate that sperm DNA derived effects may condition human embryo quality. In this respect, in a shared donor oocyte program, it was found that zygotes and embryos obtained from sperm from males with a previous history of poor zygote quality had a significantly lower quality, when compared with the control males who proved normal zygote quality in preceding cycles (12, 28 –31). Although the origin of this paternal effect needs further clarification, sperm DNA quality clearly is one of the possible candidates. The apparent association observed between embryo quality and sperm DNA fragmentation could explain the negative correlation with implantation rate. It has been suggested that poor quality embryos would have lower probabilities to implant and this is actually reflected in our study. Several reports indicate that the pregnancy rates either natural or using IUI, IVF, and ICSI procedures tend to be lower in patients with higher levels of sperm DNA fragmentation (8, 12, 13, 29, 33–37). In our case, we observed a minor increase in sperm DNA fragmentation in patients which did not achieve pregnancy, but this difference was not found to be significant, as in other studies did (14, 38). This could be due to embryo selection before transfer, so poor prognosis embryos are not chosen. In the absence of such selection, it may be possible that the pregnancy could be associated with the sperm DNA fragmentation rate. These variations in the outcome of sperm DNA fragmentation, depending on the study and the patient, could be explained by taking into account two premises. First, DNA fragmentation may not indicate homogeneous damage, quantitatively and/or qualitatively (39). Second, the outcome would be a balance between the DNA damage from the sperm and the repairability of the oocyte (40). Normal sperm DNA appears to contain a relative higher density of backFertility and Sterility姞

ground damage than certain somatic nuclei (e.g., leukocytes) (41). Nevertheless, this should be easily repaired by the oocyte. Otherwise, those sperm nuclei considered as positive for DNA fragmentation, possibly do not have the same amount of damage, within the high range. This is evidenced in the different categories of halo, and correspondent DBDFISH labeling, obtained with the SCD test (16). Those without halo and degraded nucleus would contain more damage than those without halo, and these more than those with small halo. Nevertheless, not only the amount, but the nature and/or complexity of the DNA lesions may also not be similar in the different sperm cells considered positive for DNA fragmentation, and in the different individuals. For example, sperm containing thousands of DNA double-strand breaks (DSBs), as expected after an apoptotic-like process, should not be compatible with any chromatin metabolism, implying an irreversible status (42, 43). In mammalian animals, DSBs are mainly repaired by the nonhomologous end-joining (NHEJ) pathway. Independently, this extremely high level of DSBs widely exceeds the repair capacity that, moreover, is frequently error prone (44, 45). If penetrated within the oocyte, the extremely broken chromatin should not be able to decondense, exchange protamines by histones, and replicate, which does not give rise to a pronucleus (23). A fertilization failure would be the consequence, as observed in the present study. Otherwise, if the sperm DNA damage is mainly constituted by single-strand breaks, basic sites, and/or base damages, the base excision repair (BER) and nucleotide excision repair (NER) pathways from the oocyte could be effective, with a relatively low misrepair rate, because they are theoretically not error-prone (46 – 48). The male pronucleus may be constituted, but possibly delayed with respect to the female pronucleus, due to the repair activity. This could explain the asynchrony of nucleolar precursor bodies here reported. The misrepair rate would be proportional to the damage level, so chromosomal aberrations would be generated, leading to cell arrest in the first or subsequent cell cycles (14, 37). Consequently, a lower amount of blastocysts would be developed, as we have found. If the blastocyst stage is achieved and the DNA repair was complete in amount and fidelity, the paternal genome may be normally expressed, so a pregnancy would be likely. Then, the extreme complexity resulted by the interactive effects of the diversity of qualitative and/or quantitative DNA damage from each sperm coupled with the variable DNA repair capacity of each oocyte, could explain the different results. A more definitive clarification of the possible influences of the sperm DNA fragmentation, and combined oocyte repair proficiency, would be achieved performing more controlled experiments, using animal models. The FIV and ICSI with sperm with unequivocally defined DNA lesions, on oocytes of similar quality, should be performed for a confident correlation with the possible effects in fertilization, embryo development, and pregnancy outcome. Though with less accuracy, an approach may be performed in humans, using 381

oocytes with similar good quality, from a same woman, as in donor oocyte programs. Studying the outcome after ICSI with sperm from different males, may be a situation where the influence of the female factor, is minimum at least during the in vitro development, so evidencing the male factor more confidently. Both approaches are in course in our laboratories. Essentially, two interesting details are derived from the results. First, the determination of sperm DNA fragmentation could be of special interest for patients with low gestation rates and low embryonic quality. The DNA damage could then be a possible causal factor. Second, the observation of nucleolar asynchrony in a zygote from a sperm sample with a high DNA fragmentation level could orientate to the possibility of a future low-quality blastocyst. Therefore, this could be an interesting criterion to orientate for embryo selection when the transfer occurs on day 3. In conclusion, the degree of DNA fragmentation, established using the SCD test, appears to be related to the ability of sperm to fertilize as well as the ability of the embryo to achieve the blastocyst stage until day 6. The correlation of the sperm DNA fragmentation level with embryo health appears quite promising in predicting the quality of the embryo cohort after IVF/ICSI procedures. Acknowledgments: We thank Maria J. Morata, B.S., Yolanda Márquez, B.S., Paloma Rodriguez, B.S., Pilar Campos, B.S., Carmen Blanco, B.S., Carol Rico, B.S., and Maria Pons, B.S., for their assistance with semen processing and sampling. We also thank Juan G. Alvarez, M.D., for critical review and editing of the manuscript.

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