Medicine (Baltimore). 2016 May;95(20):e3624.

Variation of DNA Fragmentation Levels During Density Gradient Sperm Selection for Assisted Reproduction Techniques: A Possible New Male Predictive Parameter of Pregnancy?

Muratori M1, Tarozzi N, Cambi M, Boni L, Iorio AL, Passaro C, Luppino B, Nadalini M, Marchiani S, Tamburrino L, Forti G, Maggi M, Baldi E, Borini A.
From the Department of Experimental, Clinical and Biomedical Sciences, Unit of Sexual Medicine and Andrology, Center of Excellence DeNothe, University of Florence (MM, MC, ALI, CP, BL, SM, LT, GF, MM, EB); Tecnobios Procreazione, Centre for Reproductive Health, Bologna (NT, MN, AB); and Clinical Trials Coordinating Center, AOU Careggi, Istituto Toscano Tumori, Florence (LB), Italy.



Predicting the outcome of IVF/ICSI is one main goal of the current research on assisted reproduction. To understand whether density gradient centrifugation (DGC), used to select sperm, can affect sperm DNA integrity and impact pregnancy rate (PR), we prospectively evaluated sperm DNA fragmentation (sDF) by TUNEL/PI, before and after DGC . SDF was studied in a cohort of 90 infertile couples the same day of IVF/ICSI treatment. After DGC, sDF increased in 41 samples (Group A, median sDF value: 29.25% [IQR:16.01-41.63] in pre- and 60.40% [IQR: 32.92-93.53] in post-DGC) and decreased in 49 (Group B, median sDF value: 18.84% [IQR:13.70-35.47] in pre- and 8.98% [IQR:6.24-15.58] in post-DGC). PR was 17.1% and 34.4% in Group A and B respectively (OR: 2.58, 95%CI: 0.95-7.04, p=0.056). After adjustment for female factor, female and male age and female BMI, the estimated OR increased to 3.12 (95% CI: 1.05-9.27, p=0.041). According to the subgroup analysis for presence/absence of female factor, heterogeneity in the association between the Group A and B and PR emerged (OR: 4.22, 95%CI:1.16-15.30 and 1.53, 95%CI:0.23-10.40, respectively for couples without, n=59, and with, n=31, female factor).

This study provides the first evidence that the DGC procedure produces an increase in sDF in about half of the subjects undergoing IVF/ICSI, who then show a much lower probability of pregnancy, raising concerns about the safety of this selection procedure. Evaluation of sDF before and after DGC configures as a possible new prognostic parameter of pregnancy outcome in IVF/ICSI. Alternative sperm selection strategies are recommended for those subjects who undergo the damage after DGC.

PMID: 27196465; DOI:10.1097/MD.0000000000003624



Couple infertility is the inability to conceive after one year of unprotected intercourse or to carry a pregnancy to live birth. Couple infertility is becoming an issue of public health as one in seven couples of reproductive age encounters problems with fertility in western societies. Infertility can be due to female or male factors or to a combination of both. In 15-20% of the couples the causes of the infertility are unknown. Factors of infertility include dysfunctions in the production of sperm or eggs, problems in functions of male or female reproductive systems, and/or hormonal and autoimmune conditions.

About 3% of the couples are treated by assisted reproductive technology (ART). ART includes Intrauterine Insemination (IUI) where sperm is inserted into the uterus through a catheter; IVF (In Vitro Fertilization), where eggs are extracted and incubated with about 100.000 spermatozoa; ICSI (IntraCytoplasmic Sperm Injection), where a motile and apparently morphologically normal single spermatozoon is selected and injected into the egg with a microscopic needle. In case of IVF and ICSI, after fertilization of the egg and once an embryo has developed, it is transferred into the uterus.



Figure 1. Scheme of density gradient centrifugation (DGC) used in our study. Semen is layered on the top of a discontinuous density gradient (composed by 40% and 80% colloidal silicon suspensions) and then centrifuged. At the end of the procedure sperm are recovered from the bottom of the tube.


Although the great advance of ART, the current pregnancy rate of couples treated by IVF/ICSI remains quite low, about 30% [1]. ART laboratories attempt to recreate the conditions of natural conception by mimicking the physiological processes required to fertilize the oocyte and to support the subsequent embryo early development. In natural reproduction, after deposition in the vagina and during the transit in the female genital tracts, spermatozoa are dramatically reduced as number and undergo important modifications [2]. Indeed, the few spermatozoa reaching the site of fertilization show better functions/structures, such as morphology, motility and maturity [3]. In addition, a complex of membrane/cellular changes occurs during capacitation, the process which renders the spermatozoa competent to fertilize the oocyte [4]. In ART laboratory the selection and the capacitation of spermatozoa are mimicked by specific selective procedures implying the incubation of spermatozoa in media containing capacitating factors [5]. One of the most popular techniques used to select sperm is density gradient centrifugation (DGC), where semen is layered on the top of a discontinuous density gradient composed by colloidal silicon suspensions (an example is shown in Figure 1) and induced to migrate though it by centrifugation. At the end of the procedure, the spermatozoa that are recovered by the most dense layer in the bottom of the tube show a better motility, morphology and maturity than spermatozoa in neat semen [6-7]. It has long been assumed that also the DNA integrity is improved by selection, however some recent studies questioned this issue. One of these studies reported that the commercial colloidal silicon used to make the gradient can be contaminated by heavy metals that are responsible for the induction of sperm DNA fragmentation (sDF) in selected spermatozoa [8]. SDF consists in single and double stranded DNA breaks in the sperm nuclei, and impacts natural and assisted reproduction negatively. Indeed, high amounts of sDF delay or prevent natural conception, increase miscarriage rate and decrease the achievement of pregnancy after ART [9]. However, no study investigated whether the damage induced by selection could affect the pregnancy outcome after ART.

Our study aimed at investigating the effect of sperm selection with DGC on sDF levels and on the pregnancy rate in couples treated by IVF/ICSI. To measure sDF we used the flow cytometric method TUNEL/PI [10], coupling the labelling of DNA breakage (with TUNEL) to the staining of sperm nuclei (with propidium iodide) to better define cytometrically the spermatozoa. Figure 2 reports typical TUNEL/PI dot plots of one subject A and one subjects B in sperm samples before and after DGC. In the day of couple treatment, we determined sDF before and after DGC in semen samples of the male partners of the couples (n=90). We found that the average sDF percentage did not change during selection, at variance with motility that increased in selected spermatozoa, as expected (Figure 3A). However, when we analyzed the single subjects, we observed that DGC induced an increase of sDF in the 45% (41 out of 90, group A) of them, whereas in the remaining 55% DNA damage decreased during selection (group B) (Figure 3B).



Figure 2. Typical TUNEL/PI dot plots from a Subject A (upper panels) and a subject B (lower panels) as determined before and after DGC. The inserts report the corrisponding negative controls used to set the quadrants in the dot plots. SDF is determined in the UR and LR quadrants. Note the increase and the decrease of sDF in, respectively, subject A and B. NC, Negative Control. LL,Low Left. LR, Low Right. UL, Upper Light. UR, Upper Right.


The two groups of couples did not differ in the main male and female parameters that might affect achievement of pregnancy (table 1) including the level of sDF before selection (Figure 3) with the exclusion of sperm concentration which was slightly lower in group A. Also the type of couple treatment (IVF or ICSI) was similarly distributed in group A and group B. We next evaluated whether there was a difference in the achievement of the clinical pregnancy in the two groups. We found that the pregnancy rate (number of pregnancy/number of couples) in group A was 50% lower than that in group B (17.1% vs 34.7%) and the statistical analysis indicated that the strength of association between subjects B and pregnancy was 2.58 greater than that found for subjects A (OR:2.58, CI:0.95-7.04). Sperm concentration did not result associated to pregnancy (OR=1.00 95% CI:0.99-1.01). Hence, the only parameter which differed between the two groups and that affected pregnancy achievement was the outcome of DGC in terms of sDF. The statistical analysis was also performed by taking into account those parameters that could affect negatively the achievement of pregnancy including the presence of female factor of infertility, the male and female age and the female BMI (Body Mass Index, an index indicating whether a subject is obese) [11-13]. After this analysis, the group B resulted even more strongly associated to pregnancy with respect to group A (OR:3.12, CI:1.05-9.27), suggesting that the greater probability to obtain pregnancy after ART of group B respect to group A is entirely due to the different outcome of DGC selection (respectively decrease and increase of sDF level). The outcome of DGC in terms of increase/decrease of sDF appears to be a novel male parameter able to predict the success of ART treatment.

A question raised by this study is which mechanism is responsible for the changes of sDF during DGC. In subjects decreasing DNA damage during selection, the elimination of dead spermatozoa occurring during DGC can be the main mechanism reducing DNA damage as most dead cells are also DNA fragmented [14]. Regarding the increase of sDF during selection, a recent paper by Aitken’ group demonstrated that the colloid silicon reagent used to make the gradient is contaminated by heavy metals and that such contamination is responsible for a localized oxidative attack to sperm DNA in turn provoking DNA breakage [8]. In such a case, however, an increase of the damage was expected in all the subjects analyzed. Hence, we hypothesize that, in subjects where selection produces an increase of DNA damage, there is a cell/chromatin vulnerability making these men more susceptible to the insult produced by DGC. Future studies are needed to elucidate the nature of such vulnerability that could represent a cause of male infertility so far undisclosed.

Another insight of this study regards the safeness of the use of DGC in ART. Future studies are necessary to verify whether alternative procedures to prepare spermatozoa do not induce DNA damage during selection even in subjects with increased susceptibility. Among alternative procedures, swim-up technique is expected to be less harmful for spermatozoa as it does not  use colloid silicon suspensions and thus it should not expose the gametes to contaminants. Prospective studies need to be designed to assess whether swim up is really safer for sperm DNA integrity in subjects A.

Importance of the study

This study reported for the first time that the sperm DNA damage induced by DGC, a popular selection procedure used to prepare spermatozoa in ART, affects pregnancy rate provoking a 50% lower probability to achieve pregnancy in the couples experiencing such damage.



Figure 3. Mean ± SD of percentage sDF (and motility as assessed in spermatozoa before and after selection, in all subjects (upper panels) and in the two groups separately (lower panels). * p<0.001, Mann-Whitney test.



Table 1. Male and female age, presence of female and male factor and semen parameters in group A and B. Data are median [IQR].

# Group A versus B; Mann-Whitney test.



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