Materials and methods

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1 RBMOnline - Vol 19. No Reproductive BioMedicine Online; on web 28 September 2009 Article Chromomycin A3 staining, sperm chromatin structure assay and hyaluronic acid binding assay as predictors for assisted reproductive outcome Martine Nijs obtained her PhD in Medical Sciences at the Free University of Brussels in Belgium. She is ESHRE accredited senior clinical embryologist. At present, she is the director of the IVF laboratory at the Genk Institute for Fertility Technology in Genk, Belgium. Her main interests are sperm quality, sperm freezing and human embryology. Dr Martine Nijs Martine Nijs 1,3, Eva Creemers 1, Annemie Cox 1, Kim Franssen 1, Mia Janssen 1, Elke Vanheusden 1, Christopher De Jonge 2, Willem Ombelet 1 1 Genk Institute for Fertility Technology (GIFT), Ziekenhuis Oost Limburg, Genk, Belgium; 2 Reproductive Medicine Centre (RMC), University of Minnesota, Minneapolis, MN, USA 3 Correspondence: martine.nijs@zol.be Abstract Functional sperm tests such as the sperm chromatin structure assay (SCSA), chromomycin A3 staining (CMA 3 ) and hyaluronic acid binding assay (HBA) have been suggested as predictive tests of fertility in vitro. This study aimed to define the clinical role of these functional parameters in assisted reproduction in a prospective cohort study. Conventional sperm diagnosis (motility, morphology and concentration) as well as SCSA, CMA 3 and HBA tests were performed on 205 semen samples [74 IVF, 94 ICSI and 37 combined IVF/intracytoplasmic sperm injection (ICSI)]. Main outcome parameters were fertilization rate, clinical pregnancy rate and take-home baby rate. The study showed that each of the three functional sperm tests was related to one or more conventional and one or more functional sperm tests, indicating that spermatozoa from patients with abnormal conventional semen parameters have a higher likelihood for multiple functional abnormalities. Only SCSA and CMA 3 staining were shown to have a limited predictive value when IVF or combined IVF/ICSI was applied. The proposed threshold value of 15% for predicting good fertilization rates and obtaining a pregnancy in IVF could only be confirmed for percent HDS (high DNA stainability in SCSA). ICSI outcome was not influenced by any of the conventional or functional sperm parameters. Keywords: CMA 3, DNA fragmentation, IVF/ICSI outcome, SCSA, sperm morphology, sperm quality Introduction Human semen analysis provides fundamental information for clinicians for an infertility diagnosis. The conventional diagnosis of a semen sample is based on a microscopic assessment of three parameters: concentration, motility and morphology analysis of the spermatozoa in a fresh semen sample. Unfortunately, it has become apparent that the reference points for this conventional sperm diagnosis as presented in the World Health Organization Manual (WHO, 1999; Menkveld, 2007) are inconclusive for a correct infertility diagnosis and possible proposed infertility treatment by the clinician (Ombelet et al., 1995; Cendeho, 2007; Lewis, 2007; Siebert et al., 2007; Lewis et al., 2008; Zini and Sigman, 2009). Moreover, other important aspects of sperm function such as nuclear maturity, DNA and chromatin normality, sperm membrane interaction with cumulus, zona pellucida and oocytes are not being assessed 671 Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 672 by the conventional three-parameter method (Cayli et al., 2003, 2004; Huszar et al., 2003; de Liu et al., 2007). Hence, they are potentially important additional parameters for the prediction of the patient s fertilizing potential. A vast increase in the knowledge on the fundamentals of sperm biology and fertilization processes has led to the development of numerous new biochemical and molecular tests like for example the sperm chromatin structure assay (SCSA), chromomycin A 3 (CMA 3 ) staining and the hyaluronic acid binding assay (HBA) (Oehninger et al., 2000, 2007; Oehninger, 2003; Gadella and Visconti, 2006; Oliva, 2006; Carrell et al., 2007; Henkel, 2007a,b; Nixon et al., 2007; Lewis et al., 2008; Nanassy and Carrell, 2008; Vjugina and Evans, 2008). SCSA is one of the tests that can detect DNA fragmentation and abnormal nuclear maturity in spermatozoa (Evenson et al., 1999, 2002; Aitken, 2006; Henkel, 2007a,b; Tarozzi et al., 2007). SCSA outcome has been reported to have a significant negative correlation with in-vivo fertilization rates (Evenson et al., 1999; Spano et al., 2000) and intrauterine insemination (IUI) (Bungum et al., 2008), as well as embryo quality in IVF cycles (Hammadeh et al., 1998; Tomlinson et al., 2001; Seli et al., 2004; Virro et al., 2004), although the SCSA outcome could not be linked to poorer fertilization rates in intracytoplasmic sperm injection (ICSI) cycles (Twigg et al., 1998). Other studies could not confirm these correlations (Larson et al., 2000; Bungum et al., 2004, 2007; Gandini et al., 2004; Lin et al., 2008), demonstrating that successful pregnancies in IVF/ICSI cycles can even be obtained using semen samples with a high degree of DNA damage. Different studies, including meta-analyses, showed an increased trend of spontaneous abortions following IVF/ICSI for patients with a higher DNA fragmentation index (DFI) and high DNA stainability (HDS) as detected by the SCSA (Virro et al., 2004; Check et al., 2005; Lin et al., 2008; Zini et al., 2008b). During spermiogenesis, histones are replaced by protamines followed by supercoiling of the DNA strands, resulting in a highly condensed sperm nucleus. In case of abnormal protamination, histones will persist in the sperm nucleus and will hamper the decondensation process in the fertilized oocyte (Nanassy and Carrell, 2008). The quality of DNA protamination, condensation and maturity can be tested by CMA 3 staining (Bianchi et al., 1993; Manicardi et al., 1995; Lolis et al., 1996). Clear relationships have been demonstrated between CMA 3 staining and sperm morphology (Bianchi et al., 1996; Franken et al., 1999; Esterhuizen et al., 2000a). Poor chromatin packaging may contribute to failure of sperm decondensation in IVF or ICSI and may subsequently result in fertilization failure (Bianchi et al., 1996; Lolis et al., 1996; Sakkas et al., 1996; Esterhuizen et al., 2000b; Iranpour et al., 2000; Nasr-Esfahani et al., 2008). Functional aberrations can also be located at the level of sperm oocyte fusion and interfere with a successful fertilization process. Spermatozoa have at least three specific hyaluronan binding proteins involved in maturation, acrosome reaction, motility, hyaluronidase activity and sperm zona binding (Kornovski et al., 1994; Hunnicutt et al., 1996; Sabeur et al., 1998; Ghosh et al., 2007). The hyaluronic acid binding assay (HBA) identifies mature spermatozoa in the semen sample that have intact acrosomes and normal morphology and that are able to bind to a molecular layer of hyaluronic acid (HA) located on a glass slide (Cayli et al., 2003; Huszar et al., 2003; Prinosilova et al., 2009). So far, only one study has focused on the predictive value of HBA and outcome in assisted reproduction: Ye et al. (2006) found HBA to be a predictor for the sperm fertilization potential in IVF, but significantly less than sperm morphology. The aim of this prospective cohort study was two-fold: first to determine a possible relationship between conventional WHO parameters (motility, concentration and sperm morphology) and the three functional parameters (SCSA, CMA 3 staining and HBA) for patients undergoing an IVF or ICSI or IVF/ICSI treatment, and second, to evaluate the possible clinical predictive value of SCSA, CMA 3 staining and HBA in combination with the conventional parameters for the outcome of the assisted reproduction treatment for the same group of patients. Main outcome parameters studied were fertilization rate, pregnancy rate and take-home baby rate. Materials and methods Patient population The prospective study was based on a cohort of 205 consecutive infertile patients undergoing IVF, ICSI or combined IVF/ICSI during a 2 year period (July 2003 to July 2005) at the Genk Institute for Fertility Technology. Patients signed an informed consent agreeing to participate in the study. Patients were allocated to either IVF or ICSI treatment according to infertility diagnosis and sperm quality (as described previously by Ombelet et al., 1997, 2003). The choice of the fertilization method was based upon the infertility diagnosis. The criteria for performing ICSI was a good motile sperm count of < spermatozoa (grade A and B; WHO, 1999) after density gradient centrifugation and/or 4% normal forms. Patients with a history of failure of 3 IUI cycles underwent a combined IVF/ICSI treatment. Routine semen analysis For ethical reasons, semen samples obtained 7 10 days before the actual oocyte collection and not on the day of oocyte retrieval, were used for the eight types of sperm analyses. Taking into account that fluctuations in outcome of sperm diagnosis on different sperm samples from the same patient have been described [for conventional and SCSA analyses (Menkveld, 2007) but not for HBA (unpublished data)], the time to the actual assisted reproductive treatment was kept to a minimum of 5 7 days. Patients produced a fresh semen sample after 2 days of abstinence. None of the men reported fever or other illnesses during the 8 weeks prior to and during the study. Semen analysis was performed according to WHO guidelines (WHO, 1999) by the same laboratory technician; semen samples were processed within 1 h of production

3 and liquefaction. Standardization of sperm analysis was ensured by participation in internal (monthly) and external quality assessment schemes (Franken and Kruger, 2007) organised by the Belgian Scientific Institute of Public Health. The following fresh semen parameters were noted: concentration, morphology and motility (grade A, B, C and D motility) and were analysed as described in detail by Nijs et al. (2009). Grade A motile spermatozoa are rapid progressive spermatozoa with a speed of 25 lm/s; grade B spermatozoa are slowly progressive with a speed of 5 24 lm/s; grade C spermatozoa are non-progressive sperm cells and grade D spermatozoa are immotile. Good motility was defined as the sum of grade A and grade B motility. Sperm morphology was scored using strict criteria (Kruger et al., 1986; Menkveld et al., 1991). Cryopreservation of semen samples Cryopreservation and thawing was performed according to the routine laboratory procedure using a glycerol glucose based procedure and described in detail by Nijs and Ombelet (2001). Thawing of semen samples for HA and CMA 3 One straw per patient was thawed by exposure to 22 C for 2 min. Straws were cleaned, opened and emptied into a 15 ml BD Falcon test tube (352095; Leuven, Belgium). Hyaluronic acid binding assay A drop of 10 ll of the frozen thawed sperm suspension was loaded on a HBA slide from MidAtlantic Diagnostics (Marlton, USA). Freezing and thawing did not alter the HA-binding properties of the spermatozoa (Nijs et al., 2009). HBA was performed according to manufacturer s guidelines and described in detail by Nijs et al. (2009). Unbound and bound motile spermatozoa were counted (total between 100 and 200 spermatozoa). The percentage of hyaluronan binding was calculated by dividing the motile bound spermatozoa by the total motile (bound and not bound) spermatozoa and multiplied by 100. Fresh semen from a normal healthy volunteer was used to analyse inter- and intra-lot and operator variations. HA analysis was performed by the same operator. The coefficients of variation for all intra- and inter-assay assessments were <12%. CMA 3 staining Preparation for and staining by CMA 3 was performed as described by Manicardi et al. (1995) and Franken et al. (1999) and performed by the same operator. Frozen semen samples were thawed as described above and washed twice in Dulbecco s Ca 2+ Mg 2+ free phosphate-buffered saline (PBS) (two parts PBS and one part frozen thawed semen) and centrifuged at 250 g for 10 min. Spermatozoa were fixed, stained with CMA 3 and subsequently analysed by fluorescence microscopy at nm wavelength (Laborlux D; Leitz, Wetzlar, Germany). A total of 250 cells were randomly evaluated per slide using the method of Franken et al. (1999), namely bright yellow fluorescence of the sperm head (chromatin packaging abnormal) and dull yellow staining (normal chromatin packaging). CMA 3 staining identifies abnormalities in chromatin packaging, since it is a guanine cytosine specific fluorochrome and competes with protamines bound to DNA. The rare cells showing ambiguous fluorescence were not considered. Freezing and thawing did not alter the CMA 3 staining patterns of the spermatozoa as tested in a pre-experimental set up (unpublished data). Fresh semen from a normal healthy volunteer was used to analyse inter- and intra-lot and operator variations. The coefficients of variation for all intraand inter-assay assessments were <12%. SCSA One straw per patient was sent in a dry liquid nitrogen shipper to the Reproductive Medicine Centre at the University of Minnesota and stored in liquid nitrogen until time of DNA chromatin testing. Straws containing semen were removed from liquid nitrogen storage and placed in a 37 C water bath until the specimen thawed. The sample was then vortexed briefly and immediately placed in crushed ice in preparation for SCSA testing. An in-house study showed that freeze thawing did not influence of the SCSA outcome (unpublished data). The procedure for SCSA testing for chromatin integrity exactly followed that of Evenson and Jost (1994). Acridine orange (AO), which intercalates into double-stranded DNA (native; normal), fluoresces green, while AO, which associates with singlestranded (denatured) DNA, fluoresces red when excited by a 488 nm light source (Darzynkiewicz et al., 1975). The extent of sperm DNA denaturation was quantified using a FACScan flow cytometer (Becton Dickinson, Westwood, MA, USA) and was expressed as the DNA fragmentation index (%DFI), which is the ratio of red to total fluorescence intensity, i.e. the level of denatured DNA over the total DNA. The SCSA also identifies a sperm fraction with high DNA stainability (%HDS). HDS spermatozoa are considered to be immature with unprocessed nuclear proteins and/or poorly condensed chromatin. The intralaboratory coefficient of variation was found to be <5% for DFI and %HDS. Patient stimulation and oocyte collection All patients underwent ovarian stimulation treatment in order to obtain multiple oocytes. Patients received an oral contraceptive pill in the cycle prior to ovarian stimulation, starting one tablet daily from cycle day 3. On day 2 or 3 of the next menstruation, vaginal ultrasound was performed to rule out ovarian cysts, and ovarian stimulation was started if hormonal oestradiol profile was below 50 pg/ml. Ovarian stimulation was started on day 3 of the cycle, using IU human menopausal gonadotrophin (HMG, Menopur; Ferring, Belgium)/day or IU recombinant FSH/day (Puregon; Schering-Plough, Belgium). On the same day an agonist (triptoreline 0.1 mg s.c., Decapeptyl; Ipsen, Belgium) was started for 7 days. From day 8 onward, vaginal ultrasound was performed on a regular 673

4 674 basis to monitor the cycle as well as measurement of serum oestradiol concentrations. The dose of HMG or recombinant FSH was adjusted if necessary. Human chorionic gonadotrophin (HCG, 5000 IU) was used to trigger ovulation once the follicle sizes were mm in diameter. Oocyte retrieval was performed 36 h after HCG injection. Cumulus oocyte complexes (COC) were cultured in sequential media from GII or GIII media supplemented with 5% human serum albumin (HSA) (Vitrolife, Goteborg, Sweden) in 5 or 6% CO 2 atmosphere depending on the type of medium used. Sperm preparation for IVF or ICSI On the day of oocyte collection, a fresh sperm sample was delivered to the laboratory after an abstinence of ejaculate of 2 days. Volume, concentration, motility and morphology were analysed as described in the sperm analysis section. The samples were then prepared for IVF or ICSI by a three-layer density gradient centrifugation (Pure Sperm; Nidacon, Gothenburg, Sweden or Sperm Grad; Vitrolife) followed by two washing steps and equilibration in sperm medium supplemented with 5% HSA (Vitrolife) at 36.5 C in a CO 2 incubator (details are described elsewhere; Nijs and Ombelet, 2004; Nijs et al., 2009). Insemination for IVF or ICSI In IVF the COC were inseminated with 100,000 grade A motile spermatozoa per ml or 500,000 grade A and B spermatozoa per ml if not enough grade A motility was obtained. For ICSI, the washed spermatozoa were placed in the middle of a flat medium drop under oil in the ICSI dish and left to swim out from the centre to the edge of the droplet during 5 10 min. From this edge, the most motile ones were collected with the ICSI pipette and subsequently placed in the middle of the PVP drop (Vitrolife). At high magnification (640) a spermatozoon was selected (on the basis of normal head, neck and tail features) from this group, immobilized and injected (Nijs and Ombelet, 2000). For the IVF/ICSI combo group, half of the oocytes were inseminated with spermatozoa and the other half of the oocytes were submitted to the ICSI procedure. Fertilization check, embryo quality scoring and embryo transfer Fertilization was checked h post-insemination; normal fertilization was defined as a metaphase II oocyte with two pronuclei. The mean proportion of fertilized oocytes per patient was calculated. Embryo quality was assessed 48, 72, 96 and 120 h after oocyte collection according to the grading system of Nijs et al. (1993a,b), including rate of development, number of cells and percentage of fragments and/or abnormalities. Embryo transfer procedure and cryopreservation Embryo transfer was performed on day 3, 4 or 5 of culture using Frydman catheters (International Medical, Brussels, Belgium). Supernumerary grade A and B embryos were cryopreserved on day 3, 4 or 5 using a propanediol sucrose slow freezing protocol. Luteal phase support consisted of daily administration of 600 mg vaginal progesterone (Utrogestan; Besins, Drogenbos, Belgium), starting on the day after oocyte retrieval. Evaluation of pregnancy and pregnancy outcome Serum HCG concentrations were measured 13 and 15 days after oocyte retrieval to detect a pregnancy. Clinical pregnancy was confirmed by ultrasound by presence of fetal sac with heart activity 5 6 weeks after embryo transfer. Progesterone was given (Utrogestan; Besins) until week 7 of the pregnancy. The implantation rate was the chance of an individual embryo to implant, as defined by the number of gestational sacs per 100 embryos transferred. Takehome baby rate was defined as the number of pregnancies resulting in a healthy baby at home per embryo transfer. The complications at delivery, birth weight and gender of the children were recorded. Statistics Statistics included the chi-squared test, two-sample t-test, z- test and logistic regression. Correlations between the percentage DFI, HDS, HBA, CMA 3, HA binding and different assisted reproduction parameters (age, sperm concentration, sperm motility, sperm morphology, fertilization rate, pregnancy rate and take-home baby rate) were evaluated by calculating Pearson s correlation coefficient (r). A P- value <0.05 was considered statistically significant. Cross tabulations were used to test the threshold values for the different sperm parameters for fertilization rate, pregnancy rate and clinical pregnancy rate and take-home baby rate. The rationale for testing threshold values for the different sperm parameters is based on previous reports in which these threshold values were defined (morphology: Esterhuizen et al., 2000b; SCSA: Evenson and Jost, 2000; CMA 3 : Esterhuizen et al., 2000b; HBA: Huszar et al., 2003). Finally, a correction for multiple testing, to take account for testing n dependent or independent hypotheses on the same set of data, was made using a false discovery rate (FDR) control. Each individual hypothesis was therefore tested at a statistical significance level, which is lower than it would be if only one hypothesis were tested (Benjamini and Hochberg, 1995). For n = 2, the new significance level becomes a = (3/4) 0.05 = and a = (11/20) 0.05 = for n = 10. Results The study population consisted of 205 men undergoing an IVF or ICSI or IVF/ICSI combined treatment, from whom semen samples were obtained. The demographic data for the patients are presented in Table 1. Thirty-one percent of the patients had a pure female pathology, 43% suffered from exclusive male infertility and 26% were of mixed infertility origin. Clinical information on the sperm parameters (concentration, motility and morphology) as well as the

5 Table 1. Demographic data on 205 assisted reproductive technology cycles divided according to type of treatment (all assisted reproduction, IVF, ICSI, combined IVF/ICSI). Values are mean (±SD; range). Overall IVF ICSI Combined IVF/ICSI Number of patients Age of women (years) 32.2 (4.1; 21) 32.5 (4.2; 21) 32.3 (4.4; 20) 31.1 (3.1; 13) Age of men (years) 35.6 (4.8; 31) 35.7 (4.2; 21) 35.9 (4.6; 20) 35.3 (4.6; 22) Sperm concentration (10 6 /ml) (32.9; 207.6) (33.9; 161) b,c (30.2; 208) b (24.6; 92) c Grade A + B (%) 51.7 (18.4; 80) 59.8 (14.3; 76) d 47 (20.4; 79) d 51.5 (17.2; 77) Morphology (%) 5.4 (2.9; 16) 6.1 (3; 16) e 4.9 (2.9; 12) e 5.2 (2.5; 11) DFI (%) 22.1 (12.2; 73.2) 18.6 (9.4; 44.4) f 24.5 (13.2; 63.6) f 23.6 (13.5; 69.4) HDS (%) 11.9 (6.9; 45.7) 10.3 (5.5; 26.6) g 13 (7.5; 34.4) g 12.8 (7.7; 42.8) HA binding a (%) 71 (20.7; 100) 71.4 (20.8; 100) 68.9 (22.3; 94) 75.3 (16.3; 80) CMA 3 (%) 19.2 (7.4; 49) 18.1 (6; 33) 20.2 (8.6; 49) 19.1 (6.4; 31) CMA 3 = chromomycin A 3 ; DFI = DNA fragmentation index; HA = hyaluronic acid; HDS = high DNA stainability. a For 22 patients (10.7%), no HA binding assay could be performed due to too low sperm count after freeze thawing. Statistics included the t-test. A P-value of <0.05 was considered statistically significant. b P < c P < d P = e P = f P = g P = outcome of the SCSA, HA binding and CMA 3 staining tests of the 205 patients is summarized in Table 1. Conventional and functional parameters Correlation analysis between the three conventional parameters showed significant correlations between concentration and morphology (r = 0.35, P < 0.001) and good motility (grade A + grade B motility) (r = 0.32, P < 0.001). Morphology was also significantly related to good motility (grade A and grade B motility) (r = 0.29, P < 0.001). None of the conventional or functional sperm parameters could be related to men s age (as tested by Pearson s correlation coefficient (r): sperm morphology (r = 0.17); good motile spermatozoa (r = 0.12); sperm concentration (r = 0.01); d) %DFI (r = 0.31); %HDS (r = 0.09); HBA (r = 0.09) and CMA 3 (r = 0.01). Each of the three functional sperm tests (SCSA, HA binding and CMA 3 staining test) was related to one or more conventional (concentration, motility and morphology) and one or more other functional sperm tests (Table 2). In summary, significant correlations were noted for percent HA binding and morphology (r = 0.15, P = 0.04) and in a negative way with %HDS (r = 0.16, P = 0.03). A significant negative correlation for %DFI and sperm morphology (r = 0.30; P < 0.001), good motility (grade A + B) (r = 0.43, P < 0.001) and concentration (r = 0.27; P < 0.001) was seen. Percent DFI was positively related to CMA 3 staining (r = 0.15; P = 0.03). Percent HDS was negatively related to sperm morphology defects (r = 0.24; P = 0.001), good motility (grade A + B) (r = 0.18; P = 0.01) and sperm concentration (r = 0.29; P < 0.001). A positive significant correlation was noted for %HDS and CMA 3 staining (r = 0.27; P < 0.001). For 22 patients (10.7%), no HA binding assay could be performed due to too low sperm count after freeze thawing. Fertilization, pregnancy rates and takehome baby rates Of the 205 patients included in the study, 74 patients underwent an IVF treatment (36.1%), 94 patients underwent an ICSI treatment (45.9%) and 37 had a combined IVF/ICSI treatment (18.0%). Data on outcome of IVF, ICSI and combined IVF/ICSI attempts are shown in Table 3. Correlation between individual sperm parameters and assisted reproduction outcome No individual sperm parameters could be related to the fertilization rate, pregnancy rate, clinical pregnancy rate, takehome baby rate or complications during pregnancy and delivery in the overall assisted reproduction group (all P > 0.05). For specific in-vitro assisted reproductive procedures such as IVF and combined IVF/ICSI, however, three significant correlations were noted: %DFI was negatively related to good fertilization rates in IVF (>50% of MII oocytes fertilized; P = 0.04) and %HDS was positively related to the pregnancy rate (P = 0.04). Sperm concentration was significantly related to pregnancy rate in the combined IVF/ICSI cases (P = 0.03) (Figure 1). All other correlations had P >

6 676 Table 2. Correlations between hyaluronic acid (HA) binding, percent DNA fragmentation index (%DFI), percent high DNA stainability (%HDS), chromomycin A 3 (CMA 3 ) and concentration, morphology and good motile spermatozoa of 205 consecutive patients entering the assisted reproductive technology programme. HA binding c %DFI %HDS CMA 3 Morphology Good motility d Concentration HA binding c r a a P NS 0.03 NS 0.04 NS NS %DFI r a 0.30 b 0.43 b 0.27 b P NS NS 0.03 <0.001 <0.001 <0.001 %HDS r 0.16 a b 0.24 b 0.18 b 0.29 b P 0.03 NS < <0.001 CMA 3 r a 0.27 b b P NS 0.03 <0.001 NS NS <0.001 Article - Predictors for assisted reproductive outcome - M Nijs et al. Morphology r 0.15 a 0.31 b 0.24 b b 0.35 b P 0.04 < NS <0.001 <0.001 Good motility d, MotA + MotB r b 0.18 a b 0.32 b P NS < NS <0.001 <0.001 Concentration r b 0.29 b 0.25 b 0.35 b 0.32 b P NS <0.001 <0.001 <0.001 <0.001 <0.001 Statistics included the Pearson s correlation coefficient (r). A P-value (2-tailed) of <0.05 was considered statistically significant. NS = not statistically significant. a Correlation is significant at the 0.05 level. b Correlation is significant at the level. c For 22 patients (10.7%), no HA binding assay could be performed due to too low sperm count after freeze thawing. d Good motility is sum of grade A and B motile spermatozoa (World Health Organization criteria).

7 Table 3. Data on outcome of IVF, ICSI and combined IVF/ICSI attempts for 205 patients. Overall IVF ICSI Combined IVF/ ICSI Number of patients Number of MII oocytes Number MII fertilized 1397/ / / /409 MP MII fertilized % 67.2 (21.9; 0 100) 69 (23.1; 0 100) 65.7 (21.7; 0 100) 66.3 (19.8; ) (±SD; range) Patients with embryo 201/205 a (98) 72/74 (97.3) 92/94 (97.9) 37/37 (100) transfer per patients initiated (%) Mean number of embryos 1.6 (0.53; 0 2) 1.56 (0.55; 0 2) 1.68 (0.53; 0 2) 1.54 (0.51; 1 2) per embryo transfer (±SD; range) Number of pregnancies 95/201 (47.2) 40/72 (55.6) 41/92 (44.6) 14/37 (37.8) per embryo transfer (%) Clinical pregnancy rate per 60/201 (29.9) 26/72 (36.1) 24/92 (26.1) 10/37 (27.0) embryo transfer (%) Take-home baby rate per 56/201 (27.9) 23/72 (31.9) 23/92 (25.0) 10/37 (27.0) embryo transfer (%) Implantation rate (%) 66/323 (20.4) 29/112 (25.9) 25/154 (16.2) 12/57 (21.1) Number of twin 6 (6.3) 3 (7.5) 1 (2.4) 2 (14.3) pregnancies (%) Number of babies born Complications (per clinical 8 (13.3) 4 (15.4) 2 (8.3) 2 (20.0) pregnancy) (%) Late miscarriage Preterm delivery 4 1 twin 1 2 Birth weight (g) [mean (range)] 3230 ( ) 3160 ( ) b 3390 ( ) 2820 ( ) Gender male to female 29/33 (0.88) 13/13 (1) 11/13 (0.85) 5/7 (0.71) MII = metaphase II; MP = mean proportion. a Two patients had no embryo transfer because of risk for ovarian hyperstimulation syndrome, all embryos were frozen (one IVF and one ICSI case). Two patients had no embryo transfer because of failure of fertilization (one IVF and one ICSI case). b Birth weight of one baby is unknown. Predictive value of sperm parameters and pregnancy rate and take-home baby rate Logistic regression showed that the combination of the eight sperm parameters could not predict the probability of having good fertilization nor the chance of having a healthy baby at home for the overall assisted reproduction group (Nagelkerke R 2 = 6.9 and 8.1% respectively).the model did, however, show that %HDS and %DFI were good probability indicators for obtaining a pregnancy by Figure 1. Box plot illustration of the significant correlations of the individual sperm parameters, percent DNA fragmentation index (DFI) with good fertilization rate in IVF, percent high DNA stainability (HDS) with pregnancy rate in IVF and sperm concentration and pregnancy rate in a combined IVF/ICSI cycle. Statistical analysis was performed using the t-test. A P-value <0.05 was considered statistically significant. (a) IVF: percent DFI is significantly related to good fertilization rates in IVF (n = 74, P = 0.04). (b) IVF: percent HDS is significantly related to pregnancy rate (n = 74, P = 0.04). (c) In combined IVF/ICSI sperm concentration is significantly related to pregnancy rate (n = 37, P = 0.03). 677

8 678 any specific assisted reproductive technique (P = 0.049; B= 0.033; P = 0.01; B = 0.08 respectively, Nagelkerke R 2 = 8.1%). However, when isolating these two parameters, this probability value to become pregnant by any assisted reproductive technique was lost for %HDS as well as %DFI (both non-significant; Nagelkerke R 2 = 2.7%). Logistic regression for IVF, ICSI and combined IVF/ICSI cycles showed again that the combination of the eight sperm parameters could not predict the probability of having good fertilization, obtaining a pregnancy or taking a healthy baby home. Percent HDS was shown to be a parameter of value in this eight parameter analysis defining the probability for good fertilization, becoming pregnant and taking a healthy baby home in IVF cycles (P = 0.01; B= 0.106, Nagelkerke R 2 = 20.5%; P = 0.01; B = 0.149, Nagelkerke R 2 = 20.5%; P = 0.02; B = 0.172, Nagelkerke R 2 = 26.5% respectively). When using a set of two DNA maturation parameters (%HDS and CMA 3 )in logistic regression, CMA 3 was significantly related to the probability of obtaining a pregnancy (in assisted reproduction and combined IVF/ICSI; (P = 0.04; B = 4.401, Nagelkerke R 2 = 5.1%; P = 0.04; B = , Nagelkerke R 2 = 23.2% respectively) and taking a healthy baby home in IVF and combined IVF/ICSI (P = 0.04; B = 9.473, Nagelkerke R 2 = 11.2%; P = 0.04; B = , Nagelkerke R 2 = 19.7% respectively; Table 4). The statistical significance of the last three findings, however, would be eliminated by correction for multiple testing of data. Analyses were also performed to test the different threshold values for predicting good fertilization, pregnancy outcome and delivery outcome as proposed by different authors for the functional and conventional sperm parameters (Table 5). No threshold value for morphology, percent DFI, HBA and CMA 3 staining in relation to good fertilization, pregnancy rate and take-home baby rate could be established. The results, however, validated the threshold of 15% for percent HDS in overall assisted reproduction. Moreover, this 15% threshold value for percent HDS was also valid for obtaining good fertilization and a pregnancy when IVF was applied. Discussion So far as is known, this is the first prospective cohort study on the predictive value of eight different morphological and functional sperm parameters in relation to the outcome of an assisted reproductive treatment: standard IVF, ICSI and combined IVF/ICSI. Three major conclusions can be drawn from this: spermatozoa from patients with abnormal conventional semen parameters have a higher chance for multiple functional abnormalities, since each of the three functional sperm tests (SCSA, CMA 3 staining and HBA) was related to one or more conventional (concentration, motility and morphology) and one or more functional sperm test. Second, only SCSA and CMA 3 staining had a limited predictive value when IVF or combined IVF/ICSI was applied. The proposed threshold value of 15% for predicting good fertilization rates and obtaining a pregnancy in IVF could only be confirmed for %HDS. Third, outcome after ICSI was not influenced by any of the Table 4. Logistic regression for the set of two sperm parameters [percent high DNA stainability (%HDS) and chromomycin A 3 (CMA 3 )] and the probability of having a pregnancy or a healthy baby at home with assisted reproductive techniques, IVF, intracytoplasmic sperm injection (ICSI) or combined IVF/ICSI. Probability of achieving pregnancy with Probability of having a healthy baby at home with Overall IVF ICSI IVF/ICSI Overall IVF ICSI IVF/ICSI Nagelkerke R 2 (%) Parameter B P B P B P B P B P B P B P B P %HDS NS NS NS NS NS NS CMA NS NS NS NS NS = not statistically significant.

9 Table 5. Testing the proposed threshold values for sperm parameters in predicting good fertilization, a clinical pregnancy or a healthy baby at home by an assisted reproductive treatment (P-value). Proposed threshold values Type of assisted reproductive treatment Good fertilization rate Pregnancy Healthy baby at home Morphology: 4%; reduced fertility; Esterhuizen et al., 2000b %DFI: 15%, 15 30%, 30%; limited fertility over 30%; Evenson and Jost (2000) Overall IVF ICSI IVF/ICSI Overall IVF ICSI IVF/ICSI %HDS: 15%; Evenson and Jost (2000) Overall IVF ICSI IVF/ICSI %CMA 3 : 30%; Esterhuizen et al. (2000b), Razavi et al. (2003) %HBA: 50%, 50 80%, 80%; 80%; normal maturity and physiological function; Huszar et al. (2003) Overall IVF ICSI IVF/ICSI Overall IVF ICSI IVF/ICSI Significance was determined using the Pearson chi-squared test. CMA 3 = chromomycin A 3 ; DFI = DNA fragmentation index; HDS = high DNA stainability; ICSI = intracytoplasmic sperm injection; Statistically significant values are shown in bold typeface. conventional or functional sperm parameters, reinforcing that the ICSI technique clearly bypasses some biological abnormalities. This study, based on a cohort of 205 assisted reproduction patients, demonstrated a significant positive correlation between HA binding and morphology of spermatozoa: morphologically normal spermatozoa have intact specific HA binding proteins that are involved in acrosome reaction, motility, hyaluronidase activity and sperm zona binding (Kornovski et al., 1994; Hunnicutt et al., 1996; Sabeur et al., 1997, 1998; Cayli et al., 2003; Ghosh et al., 2007; Prinosilova et al., 2009) and will bind to the artificial HA layer. Since even poorly motile spermatozoa show high HA binding scores, hyaluronan binding proteins that are not involved in motility, such as SPAM1 (sperm adhesion molecule; Hunnicutt et al., 1996), are probably responsible for binding to the HA molecular layer. HA binding was shown to have a significant inverse correlation with poorly condensed chromatin (tested by SCSA), but not with poor protamination (tested by CMA 3 staining). This study cannot confirm the work of Huszar et al. (2003), who postulated that only spermatozoa that have completed the spermiogenetic processes including nuclear histone protamine replacement would bind to the HA layer. Hence, the presence of active hyaluronan binding protein(s) is not a guarantee for normal nuclear maturity in human spermatozoa. Multiple studies have focused on the correlation between DNA fragmentation (as tested by SCSA) and the three conventional sperm parameters resulting in variable and conflicting outcomes for pregnancy planners, infertile patients with and without an assisted reproductive treatment (Zini et al., 2001; Evenson et al., 2002; Giwercman et al., 2003; Bungum et al., 2004; Virro et al., 2004). All studies, including the present one, indicated a clear link between at least one poor sperm parameter and increased DNA damage (Evenson et al., 1999; Saleh et al., 2003; Erenpreiss et al., 2006). Zini et al. (2008a) performed a detailed study of the different sperm morphology abnormalities and could demonstrate a strong correlation between percent HDS (sperm chromatin condensation) and sperm head defects. Since morphologically abnormal spermatozoa are one of the sources of excess reactive oxygen species (ROS), this might be one of the causes for higher levels of DNA damage found in these teratozoospermic patients (Aitken et al. 1992; Saleh et al., 2003). The data confirm these observa- 679

10 680 tions, since a clear correlation was observed between high %DFI and %HDS and poor chromatin packaging (%CMA 3 ) as well as sperm morphology (Tavalaee et al., 2008). Indeed, ROS could play a role in reduced motility (by peroxidation of the sperm membranes) and increased DNA fragmentation (a correlation was found between DNA fragmentation and motility in this study) (Gandini et al., 2000; Giwercman et al., 2003). This could influence membrane fluidity, a requisite for capacitation and acrosome reaction and hence result in lower fertilizing potential in vivo, in IUI or in IVF. Many studies have demonstrated that alterations in sperm chromosomal proteins (histones and protamines) might be connected with male infertility. Sperm from infertile patients often exhibit anomalies linked to the type and proportion of protamines present (de Yebra et al., 1993; Carrell and Liu, 2001; Nasr-Esfahani et al., 2004; Aoki et al., 2006; Oliva, 2006; Torregrosa et al., 2006; Zhang et al., 2006; Singleton et al., 2007; Gázquez et al., 2008; Zini et al., 2008b). The present prospective cohort study could identify a correlation between abnormal chromatin packaging and sperm concentration (confirming the work of others; Lolis et al., 1996; Iranpour et al., 2000). Moreover, poor chromatin packaging was linked to a higher risk for DNA fragmentation (%DFI) and poorer DNA maturity (%HDS), confirming the work of others (Tavalaee et al., 2008). Surprisingly, no correlation was observed with sperm morphology, contradicting the work of other investigators (Bianchi et al., 1996; Franken et al., 1999; Esterhuizen et al., 2000a). It must be emphasized that all foregoing studies included different non-randomized patient populations ranging from sperm donors, men enrolling for military service, pregnancy planners, patients presenting themselves in the sperm diagnostic laboratory or patients undergoing an IUI, IVF or ICSI treatment. Comparison of outcomes from these studies should therefore be made with caution. This prospective cohort sperm parameter study suggests that sperm with abnormal semen parameters can be characterized by multiple functional abnormalities. The SCSA, CMA 3 staining and HBA are therefore to be considered as complementary rather than as strongly linked tests; they could be useful adjuncts to the conventional WHO semen analysis. As in the study of Bungum et al. (2007), the present study found that ICSI was a more efficient fertilization method than IVF: although mean percent DFI was significantly higher in the ICSI group (24.5 versus 18.6%, P = 0.006), it had no influence on the ICSI outcome. ICSI was also shown to have better pregnancy and pregnancy outcome probabilities than IVF cycles for the same levels of %HDS and %CMA 3, both tests that analyse spermatozoa for sperm nuclear maturity. Perhaps as Bungum et al. (2007) postulated, women undergoing an ICSI treatment, on average, produce healthier oocytes with a better DNA repair capacity than women undergoing IVF, since in the ICSI group, infertility is mainly caused by male factor. In the present study, sperm preparation could not have been of influence, since it was identical for all three insemination techniques, the density gradient centrifugation resulted in a decrease in the fraction of DNA defective sperm initially present in the neat semen (described by many authors: Spano et al., 1999; Larson et al., 2000; Sakkas et al., 2000; Hammadeh et al., 2001; Tomlinson et al., 2001; Gandini et al., 2004; Morrell et al., 2004; Bungum et al., 2008). It is, however, possible that specific culture medium factors influenced the outcome for IVF differently then for ICSI: while IVF oocytes were exposed for up to 16 h to the sperm suspension, in ICSI the spermatozoon was injected directly into the oocyte. In ICSI the oocyte could therefore be less exposed to ROS than in IVF. Moreover, PVP used during the ICSI procedure has been described as a scavenger for ROS. The only difference between the two insemination techniques is the extra sperm selection procedure prior to ICSI that has probably resulted in selecting spermatozoa with low DNA fragmentation and good chromatin condensation and protamination. Two recent independent metaanalyses on the predictive value of SCSA in assisted reproduction (Collins et al., 2008; Zini and Sigman, 2009; Zini et al., 2008c) showed an increased risk of embryonic loss in pregnancies achieved by the use of semen with high rates of DNA breaks as measured by SCSA in IVF and ICSI. In the present study this correlation is rather limited, since only %HDS and %CMA 3 (and not %DFI) were shown to be of predictive value for early pregnancy loss in IVF and only when the outcome of two or eight sperm parameters was combined in logistic regression. Other investigators have demonstrated decreased fertilization and embryo development to be associated with poor DNA protamination (Lolis et al., 1996; Esterhuizen et al., 2000a,b; Benchaib et al., 2003; Razavi et al., 2003; Nasr-Esfahani et al., 2005, 2007, 2008; Bakos et al., 2008). Patients with a history of failure of at least three IUI cycles could have a sperm oocyte recognition or fusion problem, and therefore a combined IVF/ICSI treatment was proposed as first line treatment. In this way, potential total failure of fertilization is avoided when performing exclusively IVF in a first cycle. Since high levels of sperm DNA fragmentation have generally been associated with lower IUI pregnancy rates (Duran et al., 2002; Evenson and Wixon, 2006; Muriel et al., 2006; Bungum et al., 2007), the failure in IUI population would be expected to have significantly higher DNA damage levels, but this was not the case. Only the sperm concentration was related to the pregnancy outcome for the combined IVF/ICSI group, probably because patients with better sperm concentrations had already achieved a pregnancy in the previous IUI treatments. Poor DNA protamination (CMA 3 ) when combined with percent HDS was related to a lower chance of pregnancy and more early miscarriages in the combined IVF/ICSI group. Both tests analyse two aspects of nuclear maturation in spermatozoa: correct protamination of sperm DNA as well as disulphide cross-linking between adjacent protamine molecules during chromatin condensation. As this study indicates, a poor sperm nuclear maturation status can give rise to lower implantation and higher early miscarriage rates. Hence, it can be assumed that these chromatin disorders in sub-fertile and infertile spermatozoa are the indirect results of a more severe disturbance of genetic material.

11 Surprisingly, no diagnostic or clinical role could be established for the HBA in IVF or the combined IVF/ICSI group, since especially in the latter group it was suspected that sperm recognition and fusion was one of the possible causes for their failure in IUI. So far, only one study has addressed the clinical value of HBA in IVF cycles (Ye et al., 2006) and could only demonstrate a limited value for diagnosis and management of male infertility. The role of the sperm receptors tested by HBA has to be further investigated. This study could not demonstrate any significant differences in delivery outcome in terms of late miscarriage or health problems in the babies born by IVF, ICSI or combined IVF/ICSI technique, nor could any correlation or predictive value be established with any of the conventional or functional sperm parameters to these outcomes. This study is the first to evaluate the diagnostic and clinical role of different functional parameters (DNA fragmentation, sperm protamination and compaction and condensation, sperm hyaluron binding) and conventional WHO sperm parameters in relation to the outcome of assisted reproduction. In summary, the study confirms that spermatozoa from patients with abnormal conventional semen parameters have a higher likelihood for multiple functional abnormalities. Sperm nuclear maturation proved to be a parameter with some predictive importance since %HDS (in SCSA) could be used as a single parameter, or in combination with CMA 3 outcome as a predictor of pregnancy and birth in couples undergoing IVF. Furthermore, it was possible to confirm the threshold value of 15% HDS for obtaining fertilization and a pregnancy when IVF was the mode of insemination. The clinical value of %DFI was showed to be rather limited, since as a single parameter it was only related to good fertilization rates in IVF. Although related to sperm nuclear condensation status and sperm morphology, no predictive role could be attributed to HBA. It was shown that the outcome of ICSI was not influenced by any of the conventional or functional sperm parameters, demonstrating that ICSI clearly bypasses biological abnormalities. Acknowledgements Thanks are due to Marie Lafromboise from the Reproductive Medicine Centre of the University of Minnesota, USA for her excellent laboratory work on the SCSA and Pieter van Gelder, PhD, from PSCT B.V. in The Hague, The Netherlands for his collaboration in the statistical analysis. References Aitken R 2006 Sperm function test and fertility. 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