ORIGINAL ARTICLE: ANDROLOGY

Are sperm DNA fragmentation,hyperactivation, andhyaluronan-binding ability predictivefor fertilization and embryodevelopment in in vitro fertilizationand intracytoplasmicsperm injection?

Barbara Pregl Breznik, B.Sc., Borut Kova�ci�c, Ph.D., and Veljko Vlaisavljevi�c, Ph.D., M.D.

Department of Reproductive Medicine and Gynaecologic Endocrinology, University Medical Centre, Maribor, Slovenia

Objective: To determine the diagnostic value of the following sperm function tests in predicting the fertilizing ability of spermatozoa inconventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI): hyaluronan-binding assay (HBA), DNAfragmentation (Halosperm), and hyperactivity.Design: Prospective study.Setting: University medical center.Patient(s): 133 couples undergoing infertility treatment with IVF/ICSI.Intervention(s): Analysis of sperm DNA fragmentation, hyaluronan-binding ability, and hyperactivation on washed semen samplesused for the insemination of oocytes.Main Outcome Measure(s): Correlation between the results of sperm function tests and the fertilization rate (FR) or embryo quality(EQ) after IVF and ICSI. Comparison of the sperm DNA fragmentation, hyperactivation, and hyaluronan binding ability between cycleswith less than 50% (group 1) and more than 50% (group 2) of oocytes fertilized after IVF.Result(s): Both FR and EQ in IVF cycles negatively correlated with sperm DNA fragmentation. Furthermore, a positive correlation wasobserved between FR and hyaluronan-binding ability or induced hyperactivity. The semen samples from the IVF cycles with low FR(group 1) were characterized by statistically significantly higher sperm DNA fragmentation and lower hyaluronan-binding ability incomparison with semen samples from the group with high levels of fertilization (group 2). In ICSI cycles, no relationship was foundbetween sperm function tests and FR or EQ.

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Conclusion(s): The Halosperm test, the HBA test, and induced hyperactivity are useful in pre-dicting the ability of spermatozoa to fertilize oocytes in IVF and are helpful in distinguishingsemen samples suitable for IVF or ICSI. (Fertil Steril� 2013;-:-–-.�2013 by American So-ciety for Reproductive Medicine.)Key Words: Blastocyst, fertilization, halosperm, hyaluronan-binding assay, hyperactivity

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ompared with conventional

Received August 11, 2012; revised November 22, 2012; accepted November 26, 2012.B.P.B. has nothing to disclose. B.K. has nothing to disclose. V.V. has nothing to disclose.Reprint requests: Barbara Pregl Breznik, B.Sc., Department of Reproductive Medicine and Gynaeco-

logic Endocrinology, University Medical Centre Maribor, Ljubljanska ulica 5, 2000 Maribor, Slov-enia (E-mail: breznik.barbara@siol.net).

Fertility and Sterility® Vol. -, No. -, - 2013 0015-0282/$36.00Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc.http://dx.doi.org/10.1016/j.fertnstert.2012.11.048

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C invitro fertilization (IVF), intracy-toplasmic sperm injection (ICSI)

ensures a higher fertilization rate (1–3),especially in patients with poor semensamples (4). Furthermore, ICSI morefrequently avoids total fertilizationfailure (TFF) (5, 6). However, ICSI is

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ORIGINAL ARTICLE: ANDROLOGY

a more invasive insemination method than conventional IVFand bypasses natural selection of spermatozoa. Using ICSI forinseminating oocytes without a clear indication can lead toeven lower fertilization and implantation rates compared withconventional IVF (7–11). Despite this stipulation, the EuropeanIVF-monitoring program (EIM) for 2010 reported that the ICSImethod for oocyte insemination was used in almost 70% of allmedically assisted reproduction cycles (12).

Many infertility clinicsmake a decision to use either the IVFor the ICSI method for oocyte insemination on the basis of clas-sic semen analysis, taking into consideration the concentration,motility, and morphology of the spermatozoa. However, the di-agnostic sensitivity of such assessment is low, and male factorinfertility is frequentlyundetectedwhenonly these threeparam-eters are considered (13–17). Approximately 15% of males withproven male factor infertility have normal spermiograms (18).

Accordingly, various additional laboratory tests havebeen developed to assess the functionality of the spermatozoa.Among them, only the tests for the hyaluronan-binding abil-ity (HBA), sperm DNA fragmentation (Halosperm), and hyper-activation of the spermatozoa are simple and quick enoughthat the same semen sample tested can be used for the insem-ination of oocytes.

Spermatozoa that are unable to bind to hyaluronan exhibitmany aspects of immaturity. They retain cytoplasm on thesperm neck and histones in the nucleus, show higher aberrantsperm head morphology, and have lower genomic integrity(19, 20). It has also been proven that sperm DNAfragmentation is associated with male infertility, correlatingwith the motility (21–23) and morphology of spermatozoa(24). Furthermore, some investigators have suggested thatsperm DNA fragmentation could compromise hyaluronan-binding ability (23, 25) as well as ability to penetrate the zonapellucida and to hyperactivate (26). Obviously, sperm DNAfragmentation compromisesmany vital functions of the sperm.

Many studies have evaluated the relationship between theresults of sperm function tests and the medically assisted re-production outcome (22–24, 27–36). However, the results ofthe studies are contradictory. A meta-analysis of these studieshas shown that sperm DNA fragmentation is only definitelyassociated with the pregnancy rate and abortion rate(37, 38). In most of the studies, the analyses were performedon native or frozen-thawed semen samples or even monthsbefore infertility treatment.

We evaluated whether the HBA, spontaneous and in-duced hyperactivation of spermatozoa, or sperm DNA frag-mentation (assessed with a Halosperm kit) can give anyadditional information about the ability of the spermatozoato fertilize oocytes in conventional IVF and to distinguish be-tween semen samples suitable for IVF and those for ICSI.Sperm function tests were performed on washed semen sam-ples that were simultaneously used for oocyte inseminationwith IVF and ICSI.

MATERIALS AND METHODSThe study was approved by the national medical ethics com-mittee, and additional analyses on sperm samples were per-formedwhenwritten consent frompatients had been obtained.

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Patients

The study included 133 couples having their first or secondinfertility treatment with IVF-ICSI. The women were underthe age of 37 years and did not have endometriosis, polycysticovaries (PCO), or polycystic ovary syndrome (PCOS). We in-cluded couples with previous diagnosed mild male factor in-fertility (concentration of spermatozoa in native ejaculatewas more than 5 � 106/mL and less than 15 � 106/mL andpercentage of motile spermatozoa was more than 25% andless than 40%), couples with several unsuccessful intrauterineinseminations, and couples with idiopathic infertility. Onlythe couples with at least six oocytes retrieved after follicularaspiration were included in the study.

Stimulation Protocols

The stimulation protocols included a combination ofgonadotropin-releasing hormone (GnRH) agonist/GnRHantagonist and recombinant follicle-stimulating hormone/human menopausal gonadotropin (FSH/hMG). Human cho-rionic gonadotropin (hCG) was administered in patients forwhom the average follicular diameter was >18 mm. A follic-ular puncture and oocyte pickup was performed 35 to 36hours after the hCG injection.

Semen Analysis

Semen samples were obtained by masturbation after 2 to 5days of sexual abstinence. After the liquefaction of the semen,the samples were examined for concentration, motility, andmorphology, according to World Health Organization (WHO)guidelines (39). The sperm concentration in native and washedsemen samples was determined with an improved Neubauerhemocytometer, according to the 2010 WHO guidelines.

Spermmotility innative andwashed semensampleswas as-sessed with the Sperm Class Analyzer (SCA; Microoptics) usingthe SCA 5.0 software version. The analysis was performed in 20mm–deep Leja two-chamber slides, according to 2010 WHOguidelines (39). Each chamber was filled with a 6.5 mL semensample. The tracks of at least 200 motile spermatozoa were an-alyzed in six representativefields per chamber (12fields in total).

The morphology of sperm from native semen was evalu-ated by Tygerberg's strict criteria, and the slides were stainedusing the Papanicolaou technique. The morphologic charac-teristics of the washed spermatozoa were assessed usingCell-vu prestained slides (Millennium Sciences, Inc.).

Semen Preparation

Motile sperm were selected using the colloidal density gradi-ent with layers of 2 mL of 80% and 2 mL of 40% Puresperm(Nidacon International AB). The entire volume of semenwas gently added to the top layer of the gradient, and thetubes were centrifuged at 600 � g for 20 minutes. The super-natant was discarded, and the pellet was resuspended with3 mL of sperm preparation media (Origio) and centrifuged at600� g for 10 minutes. The supernatant was again discarded,and the pellet was overlaid with 0.5 mL of fresh medium, al-lowing the spermatozoa to swim up (at 37�C, for 30 minutes).

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The upper layer was separated, and the prepared semen sam-ples were used for inseminating oocytes using the IVF andICSI methods and for further analyses.

Analyses on Prepared Semen Samples

Determination of DNA fragmentationwas performed using theHalosperm kit (INDAS Laboratories). A sample aliquot wastaken from each washed sperm sample. Gelled aliquots oflow-melting-point agarose in an Eppendorf tube were placedin a microwave for 4 minutes to fuse the agarose and werethen placed in a water bath at 37�C. After 5 minutes of incuba-tion for temperature equilibration at 37�C, 25 mL of the semensample was added to the Eppendorf tube and mixed with thefused agarose. Twenty-five microliters of the semen-agarosemixture was pipetted onto precoated slides and covered witha 22� 22 mm coverslip. The slides were placed on a cold plate(4�C) for 5 minutes to allow the agarose to produce a microgelwith the sperm cells embedded within it. The coverslips wereremoved and immersed horizontally in an acid solution thathad been previously prepared by mixing 80 mL of HCl froman Eppendorf tube with 10 mL of distilled water and incubatedfor 7 minutes. The slides were horizontally immersed in 10 mLof the lysing solution for 25 minutes. After washing the slidesfor 5 minutes with an abundant volume of distilled water, thesamples were dehydrated in increasing concentrations of eth-anol (70%, 90%, 100%) for 2 minutes each, air-dried, andstored at room temperature in opaque closed boxes.

For bright-field microscopy, the slides were horizontallycovered with a mix of Wright's staining solution (Merck)and a phosphate-buffered solution (1:1; Merck) for 20 to 25minutes with continuous airflow. The slides were then brieflywashed in water and allowed to dry. A minimum of 500 sper-matozoa per sample were scored under the 200 objectives ofthe microscope (40). Sperm cells with very small halos andwithout halos, as well as degraded sperm cells, were evaluatedas containing fragmented DNA (41, 42).

The analysis of hyperactivation was performed using theSCA as previously described. The following motion parameterswere measured by the SCA system and used for statistical anal-ysis: lateral head displacement (ALH), beat cross frequency(BCF), curvilinear velocity (VCL), straight linevelocity (VSL), av-erage path velocity (VAP), and linearity (LIN, ratio of VSL/VCL).The hyperactivated spermatozoa were defined by the followingcriteria: ALHR5.0 mm, LIN%60%, and VCLR100 mm/s (43).

We calculated the percentage of spontaneous and in-duced hyperactivated spermatozoa in the washed semen sam-ples. Hyperactivation was induced with HAmax reagent. TheHAmax reagent was freshly prepared daily from 2 mL ofsperm preparation medium (SPM), 40 mg of bovine serum al-bumin (BSA), and 4 mL of a stock progesterone solution (1 mgof progesterone [Sigma] diluted in 1 mL of absolute ethanol).To induce hyperactivation, an aliquot of washed semen sam-ple was mixed with the HAmax reagent in a 1:1 ratio. To con-trol for spontaneous hyperactivation, an aliquot of washedsemen sample was mixed with SPM.We incubated these sam-ples for 1 hour at 37�C in an atmosphere with 6% CO2 (44).After 1 hour, we analyzed both samples for the percentageof hyperactivated spermatozoa as described earlier.

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The percentage of spermatozoa with the ability to bind tohyaluronan (HBA; Biocoat) was determined following themanufacturer's instructions. Ten microliters of semen wasadded to the HBA chamber, and the Cell-Vu grid cover slipwas attached. A cover slip provides a grid of 100 squareswithin a viewing circle. After incubating the slide for 15 min-utes, we counted the unbound motile sperm and the boundmotile spermatozoa, and at least 200 spermatozoa in thesame square or the entire 100 squares were counted. The per-centage of spermatozoa binding to the hyaluronan was calcu-lated by dividing the number of motile bound spermatozoa bythe sum of motile bound and unbound spermatozoa.

Embryo Culture, Fertilization, and EmbryoAssessment

Oocytes and embryos were cultured in BlastAssist Systemme-dia (Origio) in an atmosphere of 5% O2, 6% CO2, and 89% N2

for a maximum of 6 days after oocyte pickup. One-half of theoocytes retrieved from the same patient were inseminated us-ing the ICSI method, and the other half using the conventionalIVF method (sibling oocytes). Fertilization was assessed 16 to20 hours after insemination by observing the presence of twopronuclei. A day-3 embryo was considered optimal if it con-sisted of at least eight cells without multinucleation and hadless than 20% fragments.

Blastocystswere scoredonday5according toourblastocystscoring system (45). The optimal blastocyst was an expandedblastocyst in which the blastocoel completely filled the embryo.The intracellular mass (ICM) was round or oval with manytightly packed cells. The trophoectoderm (TE) was made up ofmany ellipsoid cells, forming a cohesive epithelium. Therewere no excluded blastomeres or cytoplasmic fragments in theperivitelline space around the ICM or between TE cells (45).

Statistical Analysis

To analyze the correlations between semen characteristics andthe fertilization rate or embryo quality, we used a linear regres-sion. Student's t test for paired samples was used to test the dif-ferences in embryo quality after the IVF and ICSI methods. Weused Student's t test for unpaired samples to test the differencesin semenparameters between the twogroups divided accordingto fertilization rate (more or less than 50%). A ROC (receiver op-erating characteristic) analysis was used to set the cutoff valuesfor sperm function tests for predicting fertilization and embryodevelopment in conventional IVF. A statistical analysis wasperformed using the SPSS (SPSS Inc.) software program.

RESULTSThe mean age of men included in the study was 34.1 � 4.4years, and that of women was 32.1 � 3.2 years. The meanbody mass index (calculated as kg/m2) was 22.1 � 3.3. Themean number of ampules of gonadotropins used for ovarianstimulation was 23.8� 6.5. The etiology of infertility was un-explained in 23 couples (17.3%), and several unsuccessful in-trauterine inseminations took place in 20 couples (15%).Fifty-four couples (40.6%) had tubal factor infertility,five cou-ples (3.8%) had uterine factor infertility, and three couples(2.3%) had a combination of tubal and uterine factor infertility.

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TABLE 1

Semen characteristics of native and washed semen samples and results of function tests.

Native semen Washed semen

Semen volume (mL) 3.4 � 1.5 (0.9–9.5) –

Sperm concentration (�106/mL) 59.8 � 47.9 (5.4–261) 6.5 � 1.7 (2.2–11.4)Progressive motility (%) 33.9 � 13.2 (7.8–74.1) 89.4 � 13.3 (15.8–99.7)Normal morphology (%) 6.2 � 4.5 (0–21) 7.1 � 3.2 (1–16)Hyaluronan-binding ability (%) – 91.3 � 10.9 (15–100)Spontaneous hyperactivation (%) – 15.6 � 12.4 (0–82.5)Induced hyperactivation (%) – 57.9 � 22.5 (0–100)Sperm DNA fragmentation (%) – 3.9 � 3.6 (0.2–15.6)Note: Values are mean � standard deviation (min–max).

Pregl Breznik. Sperm function tests in IVF or ICSI. Fertil Steril 2013.

ORIGINAL ARTICLE: ANDROLOGY

The semen characteristics of native and washed semensamples are represented in Table 1. A total of 1,760 oocyteswere included in the study, 883 for IVF and 878 for ICSI.The mean number of oocytes retrieved per patient was13.23 � 6.9 (mean � standard deviation [SD]). After conven-tional IVF, 68.7% � 27.6% (mean � SD) of oocytes were fer-tilized (fertilized oocytes per oocytes inseminated with IVF).The mean percentage of metaphase II (MII) oocytes for ICSIwas 86.2% � 16.9% (mean � SD). After ICSI, 80.9% �20.6% (mean � SD) of oocytes were fertilized (fertilized oo-cytes per MII oocytes). The percentage of optimal day-3 em-bryos was statistically significantly higher after IVF thanafter ICSI (mean � standard error of the mean [SEM]:41.0% � 3.0% vs. 27.9% � 2.7%, respectively; P¼ .000).The blastocyst development rate was higher in IVF comparedwith ICSI (mean � SEM: IVF 53.0% � 2.9% vs. ICSI 35.2% �2.9%; P¼ .000). Furthermore, a statistically significantlyhigher percentage of optimal blastocysts developed afterIVF than after ICSI (mean � SEM: IVF 23.3% � 3.0% vs.ICSI 15.7% � 2.8%, respectively; P¼ .034) (sibling oocytes).

Correlations between Semen Characteristics andFertilization Rate or Embryo Quality

Correlations between semen characteristics and the fertiliza-tion rate and embryo development after IVF and ICSI are pre-sented in Table 2 and Figure 1. The couples were divided intotwo groups according to their fertilization rates after IVF:group 1, fertilization rate <50%; and group 2, fertilizationrate R50%. The groups did not differ in the mean age ofthe women (mean � SEM: 31.5 � 0.6 vs. 32.3 � 0.3; P =.248), the number of oocytes retrieved (12.5 � 1.4 vs. 13.4� 0.7; P = .510), the ampules of gonadotropins used (23.5� 1.3 vs. 23.9 � 0.6; P = .773), or the body mass index(22.3 � 0.6 vs. 22.1 � 0.3; P = .694). We found a statisticallysignificant difference between the groups in the percentage ofspermatozoa with the ability to bind to hyaluronan and in thepercentage of DNA fragmentation (Table 3).

Receiver Operating Characteristic Curves (ROC)and Function Tests

The results of the HBA and Halosperm tests were analyzed byROC curves with respect to achieving more or less than a 50%

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fertilization rate after conventional IVF. The curves werestatistically significant. We set the cutoff value for theHBA test at 90.85%, with 75% sensitivity and 55.2% specific-ity (AUCROC [area under curve] (95% confidence interval[CI]) ¼ 0.721 (0.618–0.823), P¼ .000). The cutoff value forthe Halosperm test was set at 6.45%, with 88.5% sensitivityand 44.8% specificity (AUCROC [CI] ¼ 0.664 [0.538–0.790],P¼ .007). Although induced hyperactivation statistically sig-nificantly correlated with fertilization rate after IVF, the anal-ysis of the ROC curve did not show any statistically significantdata (AUCROC [CI] ¼ 0.542 [0.435–0.649], P¼ .477).

The cycles with day-5 cultivation were included in furtheranalysis. They were divided into two groups. Group 1 con-sisted of cycles with total embryo development arrest(n ¼ 15). The cycles with at least one developed blastocystcomposed group 2 (n ¼ 97). Neither group differed in themean age of women (mean � SEM: 32.5 � 0.3 vs. 32.1 �0.9, respectively; P¼ .639) or in oocytes retrieved (mean �SEM: 10.5 � 1.1 vs. 13.2 � 0.5, respectively; P¼ .089). Thedifference in DNA fragmentation between groups 1 and 2was statistically significant (mean � SEM: 6.5 � 1.1 vs. 3.2� 0.3; P¼ .009). The cutoff value for predicting blastocyst de-velopment using the Halosperm test is 3.55%, with sensitivityof 75.3% and specificity of 73.3% (AUCROC [CI] ¼ 0.771[0.643–0.899], P¼ .001).

DISCUSSIONOur results on the negative correlation between the Halo-sperm test and the fertilization rate after IVF confirm somepreviously published findings (27, 28). However, someinvestigators did not achieve similar results (35, 37). Thefrequent occurrence of fertilization failure after IVFresulting from semen samples with a high percentage ofspermatozoa with fragmented DNA suggests thatspermatozoa compromised in this way may have difficultyin developing the pronuclear stage (36). Sakkas et al. (46)similarly observed that damaged sperm chromatin is notable to decondense, exchange protamines with histones, orreplicate, which results in fertilization failure in highproportions. They suggested that the outcome of spermpenetration into an oocyte depends on the amount and typeof DNA damage and the ability of the oocyte to repair thisdamage. The male pronucleus may be constituted, but DNA

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TABLE 2

Correlations between semen characteristics and fertilization rate and embryo development after IVF and ICSI.

IVF ICSI

Fertilizationrate

Optimalday-3

embryos

Blastocystdevelopment

rateOptimal

blastocystsFertilization

rate

Optimalday-3

embryos

Blastocystdevelopment

rateOptimal

blastocysts

Native semenConcentration

R 0.166 0.046 0.141 0.154 0.109 0.066 0.045 0.018P value .056 .597 .106 .077 .211 .448 .610 .835

MotilityR 0.145 0.092 0.135 0.149 0.052 0.004 0.048 0.097P value .095 .291 .120 .087 .552 .959 .587 .267

Normal morphologyR 0.164 0.018 0.125 0.145 0.105 0.148 0.015 0.151P value .059 .839 .150 .095 .231 .090 .864 .083

Washed semenHyaluronan-binding ability

R 0.321 0.092 0.095 0.112 0.114 0.036 0.117 0.005P value .000a .294 .278 .198 .190 .679 .179 .954

Spontaneous hyperactivationR 0.095 0.167 0.057 0.168 0.017 0.073 0.118 0.098P value .274 .053 .513 .052 .848 .402 .177 .261

Induced hyperactivationR 0.189 0.090 0.045 0.155 0.137 0.045 0.031 0.010P value .029a .300 .603 .074 .115 .610 .727 .908

Sperm DNA fragmentationR �0.436 �0.242 �0.312 �0.310 �0.037 �0.049 �0.065 �0.026P value .000a .005a .001a .001a .672 .573 .455 .766

Note: Fertilization rate after IVF is calculated per oocyte inseminated with conventional IVF method, fertilization rate after intracytoplasmic sperm injection (ICSI) in calculated per metaphase II (MII)oocyte inseminated with ICSI method.a Significant linear relationship, P< .05, R ¼ regression coefficient.

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repair can cause a delay with respect to the female pronucleusdue to the repair activity (47).

The fertilization rate after conventional IVF also corre-lated with the hyaluronan-binding ability of washed sperma-tozoa in our study. Hyaluronan is a major constituent of thecumulus oophorus matrix and may play a critical role in theselection of functionally competent spermatozoa during IVF(19, 20, 48). The association between the binding ability ofspermatozoa to hyaluronan from native ejaculate and thefertilization rate after IVF has been observed previously(49); our study concurred. However, our results wereobtained by testing washed spermatozoa.

Our study indicates that fertilization in conventional IVFcan be predicted by the proportion of induced hyperactivatedsperm in the insemination medium. In contrast, neither spon-taneous hyperactivation nor any other sperm movementparameter showed any predictive ability in IVF. Hyperactiva-tion as a part of sperm capacitation occurs after removingthe seminal plasma and sperm washing (50, 51).Spontaneous sperm hyperactivation varies widely over time,and spermatozoa do not remain in the hyperactivated stateindefinitely. Instead, they frequently switch betweenhyperactivated and nonhyperactivated movements (52).When testing the hyperactivation ability of sperm samplesafter 1 hour of incubation under capacitating conditions,only 30% to 40% of spermatozoa will reach spontaneoushyperactivation (53). In this process, the spermatozoa withgreater propulsion for reaching the oocyte or for passing

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through the cumulus oophorus and penetrating the zonapellucida are provided (50, 51). However, not allspermatozoa are able to hyperactivate, even after theartificial induction of sperm movement by twohyperactivation agonists, progesterone and pentoxifylline,to induce the maximum level of hyperactivation (53, 54).For all of these reasons, only induced, and not spontaneous,hyperactivation in washed semen represents a valuablesperm parameter.

A common dilemma for couples with borderline factor ofmale infertility is whether to use IVF or ICSI for the insemina-tion of oocytes. We proved that by extending the semen anal-ysis with three more tests, we could successfully choose theoptimal method of insemination, yielding an acceptable(i.e., greater than 50%) fertilization rate.

One of the goals of our study was to determine whetherthe observed sperm function parameters have any impacton embryo development. Of all three sperm function tests,only the results of sperm DNA fragmentation negativelycorrelated with the blastocyst development rate after conven-tional IVF, which again concurs with some other trials(28, 31). This result can be explained by the fact that theability of sperm to bind to hyaluronan and to hyperactivateis needed only for the sperm to pass through the cumulusoophorus and the zona pellucida, not for furtherdevelopment of the embryo. If the oocyte was fertilized bya spermatozoon with damaged DNA, its furtherdevelopment depended on the extent of the sperm DNA

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FIGURE 1

The correlation between the sperm DNA fragmentation and the fertilization rate (A) and the embryo development after IVF (B–D), and thecorrelation between the hyaluronan-binding ability (E) or the induced hyperactivation (F) and the fertilization rate after IVF.Pregl Breznik. Sperm function tests in IVF or ICSI. Fertil Steril 2013.

ORIGINAL ARTICLE: ANDROLOGY

damage and the ability of the oocyte to repair this damage. Ifthe damage was not reparable, development would bearrested in the first or subsequent cell cycles (47). If theblastocyst stage is achieved and the sperm DNA repair iscomplete, the paternal genome may be normally expressed.

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The lower number of blastocysts that developed in coupleswith fragmented sperm DNA in our study was thereforeexpected.

We set the cutoff value for the level of DNA fragmenta-tion of spermatozoa in washed semen samples to predict the

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TABLE 3

Differences in semen characteristics between groups of cycles with less or more than 50% of fertilization rate after conventional IVF.

Group 1Fertilization <50%

(n [ 29)

Group 2Fertilization R50%

(n [ 104) P value

Native semenConcentration (�106/mL) 44.1 � 8.5 64.2 � 4.7 .045Progressive motility (%) 30 � 2.1 35.1 � 1.3 NS

Washed semenConcentration (�106/mL) 6.6 � 0.4 6.5 � 0.14 NSProgressive motility (%) 80.7 � 3.9 91.8 � 0.9 .01HBA (%) 85.1 � 3.1 93 � 0.8 .019Sperm DNA fragmentation (%) 6.3 � 0.9 3.3 � 0.27 .004Spontaneous hyperactivation (%) 13 � 1.6 16.3 � 1.3 NSInduced hyperactivation (%) 51.6 � 4.6 59.7 � 2.1 NS

Note: Values are mean � standard error of the mean. HBA ¼ hyaluronan-binding assay; NS ¼ not statistically significant.

Pregl Breznik. Sperm function tests in IVF or ICSI. Fertil Steril 2013.

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embryo development to the blastocyst stage at 3.55%, witha sensitivity of 75.3% and a specificity of 73.3%. With thisvalue, at least one of the embryos will achieve the blastocyststage, which could be a useful tool when a decision for theshort or prolonged cultivation of embryos has to be made.

After ICSI, no sperm function tests correlated with the fer-tilization rate. These results are in line with the data fromsome studies (23, 24, 27, 29, 31, 35–37, 55) butcontradictory to the data of others (22, 28, 33, 56). Theapparent discrepancy in the studies may be a result ofdifferences in using either the native semen sample or thewashed sample for analysis. In our study, sperm DNAfragmentation, hyaluronan-binding ability, and the abilityto hyperactivate were analyzed in the same washed semensamples that were immediately used for oocyte insemination.A lack of correlation between the fertilization rate after ICSIand the results of sperm function tests can also be explainedby the fact that ICSI bypasses the natural process of fertiliza-tion, as the spermatozoon is physically deposited into theooplasm.

The correlation between embryo development and spermDNA fragmentation was also expected in the ICSI group.However, our analysis could not confirm this hypothesis.Similar results were obtained also by other investigators(27, 28, 36). An explanation for the lack of correlationcould be that the mechanical injection of a spermatozoonand polyvinylpyrrolidone (PVP) into the ooplasm influencesthe embryo development. The PVP injected along with thespermatozoon into the oocyte cannot diffuse and is notdigestible by lysosomal enzymes; it will remain in theoocyte (57). It was proven that PVP has an influence onacrosomes, mitochondria, plasma membranes, and othercellular organelles (58). Furthermore, Dozortsev et al. (59)suggested that PVP delayed the onset of calciumoscillations and sperm decondensation in the oocyte (60).Some other investigators have also reported that PVP hasan influence on the fertilization rate (61–63) and embryoquality (61–63).

Another possible reason for the lack of correlation be-tween sperm DNA fragmentation and the ICSI procedure out-

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come is the fact that the spermatozoon for injection isartificially selected. Avendano et al. (64) report that evenmor-phologically normal spermatozoa, as selected for ICSI, mayhave DNA fragmentation. However, when the DNA from themechanically selected sperm was analyzed, the correlationwith embryo development after ICSI became significant (65).

We conclude that all three function tests are useful toolsfor predicting the fertilization rate after conventional IVF.They are simple and accurate methods for the analysis ofsperm functionality. Furthermore, the results of all threefunction tests can be obtained in less than 2 hours. Theyshould be a part of the routine semen analysis in IVF becausethey are helpful whenmaking a decision whether to use IVF orICSI for oocyte insemination. The Halosperm test is also usefulfor predicting the outcomes of the embryo development.

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