ORIGINAL ARTICLE Andrology

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1 Human Reproduction, Vol.30, No.11 pp , 2015 Advanced Access publication on September 23, 2015 doi: /humrep/dev202 ORIGINAL ARTICLE Andrology Molecular karyotyping of single sperm with nuclear vacuoles identifies more chromosomal abnormalities in patients with testiculopathy than fertile controls: implications for ICSI Andrea Garolla, Barbara Sartini, Ilaria Cosci, Damiano Pizzol, Marco Ghezzi, Alessandro Bertoldo, Massimo Menegazzo, Elena Speltra, Alberto Ferlin, and Carlo Foresta* Department of Medicine, Unit of Andrology and Reproductive Medicine, University of Padova, Via Giustiniani 2, Padova 35121, Italy *Correspondence address. Tel: ; Fax: ; Submitted on March 10, 2015; resubmitted on July 27, 2015; accepted on July 30, 2015 study question: Is there a difference between molecular karyotype of single sperm selected by high-magnification microscopy from infertile patients with testicular damage and from proven fertile controls? summary answer: The molecular karyotype of single sperm from patients with testiculopathy had a significantly higher percentage of chromosomal alterations than fertile controls. what is known already: Infertile patients with testicular impairment have many sperm with aneuploidies and/or increased structural chromosome alterations. In these patients, sperm use by ICSI has poor outcome and raises concerns about the possible impact on pregnancy loss and transmission of genes abnormalities in offspring. High-magnification microscopy has been recently introduced to select morphologically better sperm aimed at improving ICSI outcome. However, there are no studies evaluating the molecular karyotype of sperm selected by this method. study design, size, duration: Three consecutive infertile patients with oligozoospermia due to testicular damage and three agematched proven fertile men attending a tertiary care center, were enrolled in the study from September to November Inclusion criteria of patients were age years, at least 2 years of infertility, oligozoospermia (sperm count below 10 million), reduced testicular volumes high FSH plasma levels and absence of altered karyotype, Y chromosome microdeletions, cystic fibrosis transmembrane conductance regulator gene mutations, sperm infections, cigarette smoking, varicocele, obesity. participants/materials, setting, methods: Participants were evaluated for sperm parameters, sex hormones and testicular color-doppler ultrasound. From each semen sample, 20 sperm with large vacuoles (LVs), 20 with small vacuoles (SVs) and 20 with no vacuoles (NVs) were retrieved individually by a micromanipulator system. Each cell was further analyzed by whole genome amplification and array comparative genomic hybridization (acgh). main results and the role of chance: The acgh allowed us to detect chromosomal aneuploidies, unbalanced translocations and complex abnormalities. Sperm selected from infertile patients showed a higher percentage of abnormal molecular karyotypes than controls (19.4 versus 7.7%, respectively, P, 0.001). In particular, sperm with LV and SV showed 38.3 and 20.0% abnormal karyotype in infertile men versus 18.3 and 5.0% in controls, respectively (both P, 0.01). Complex abnormalities were found only in the LV category. An abnormal karyotype was never found in NV sperm from both patients and controls. limitations reasons for caution: The main limitation of this study is the low number of included subjects. Moreover, a time of writing we have no data regarding the ICSI outcome using LV, SV or NV sperm. This is the first study evaluating the molecular karyotype of single sperm selected by high-magnification microscopy and further confirmation of the data is needed. wider implications of the findings: Our data showed that sperm from infertile patients with testicular impairment have a higher & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 2494 Garolla et al. percentage of abnormal molecular karyotypes than sperm from fertile controls. Therefore, if confirmed, our data suggest that the use of individually retrieved NV sperm may improve ICSI outcome in infertile men with testicular damage. study funding/competing interest(s): No external funding wassought for this study, and the authors have no conflict of interest to declare. trial registration number: N. 2401P. Key words: high-magnification microscopy / male infertility / molecular karyotype / oligozoospermia / sperm vacuoles / whole genome amplification / array comparative genomic hybridization Introduction Many conditions may lead to primary testicular failure and infertility including cryptorchidism, orchitis, testis trauma, torsions and iatrogenic forms such as gonadotoxic medications, chemo/radiotherapy, inguinal surgery and so on. It is well known that testicular impairment is frequently associated with alterations of sperm chromosomes in terms of numerical or structural abnormalities (Pang et al., 1999; Bonduelle et al., 2002). Infertile couples with male factor infertility are usually treated by ICSI, but in these cases the outcome is very poor (Krausz, 2011). Moreover, concerns have been raised in these patients about the possible transmission to progeny of ICSI of chromosomal alterations leading to congenital malformations, developmental abnormalities and a higher risk of imprinting diseases (Erenpreiss et al., 2006). The standard semen analysis is of fundamental importance to diagnose and define the severity of the male factor infertility, but it is unable to detect alterations of the sperm nucleus. In recent years, more attention has been focused on the genomic quality of the male gamete aimed at improving ICSI outcome and reducing the possible risk to transmit genetic alterations. However, current methods evaluating the sperm DNA status, such as fragmentation, nuclear condensation, aneuploidies or chromosomal structural rearrangements, are invasive for cells and thus they cannot be used to select the sperm for ICSI use. Bartoov et al. (2002) suggested a non-invasive technique of sperm selection aimed to predict the genetic normalcy of the male gamete. This technique is based on the evaluation of single sperm by high-magnification microscopy to detect the presence of minor anomalies and nuclear vacuoles, which are not visible at the standard magnification used for ICSI. Human sperm vacuoles were first described as nuclear holes when examined by electron microscopy and 2D imaging (Zamboni, 1987). Thanks to higher resolution techniques and technical progress in microscopic imaging, it was recently shown that vacuoles are not nuclear holes but concavities extending from the surface of the sperm head to the nucleus (Watanabe et al., 2009, Boitrelle et al., 2011; Perdrix and Rives, 2013). The origin and consequences of sperm head vacuoles are still subject to controversy because they have been suggested to originate from spermatogenesis impairment or abnormal maturation during male genital tract transit or acrosome modification during the acrosome reaction (Perdrix et al., 2011). The hypothesis that sperm head vacuoles originated from acrosomes has been explored by assessing vacuole parameters after induction of the acrosome reaction. A significantly decreased presence of vacuoles was observed after induction of the acrosome reaction (Kacem et al., 2010; Montjean et al., 2012). Independent of their size, vacuoles seem relatively common in the sperm heads from fertile and infertile men with normal or abnormal semen parameters (Perdrix and Rives, 2013). However, some authors observed a strong relation between the presence of large nuclear vacuoles and the impairment of sperm chromatin condensation (Franco et al., 2008; Garolla et al., 2008), a fundamental process involved in protection of the paternal genome before fertilization and in the early phases of embryonic development (Ward, 2010). Interestingly, Claussen (2005) and Finch et al. (2008) demonstrated that chromosome position in the sperm nucleus also reflects the status of chromatin integrity. Moreover, Perdrix et al. (2013) showed that nuclear position of chromosomes is different in spermatozoa with large nuclear vacuoles compared with spermatozoa with no vacuoles. These observations supported the hypothesis of an association between the presence of large nuclear vacuoles at sperm head and concomitant alterations of sperm nucleus. On this basis, some authors performed ICSI using a selection of sperm with no large vacuoles (LVs) and observed a higher fertilization, reduced abortion rate and better pregnancy outcome (Berkovitz et al., 2005; Figueira et al., 2011). However, other studies failed to observe any correlation between the presence and size of nuclear vacuoles and nuclear status. Even a recent Cochrane review comparing the ICSI and intracytoplasmic morphologically selected sperm injection (IMSI) techniques showed no significant difference in the pregnancy outcome (Teixeira et al., 2013). The aim of the present study was to evaluate the entire genome, including aneuploidies and chromosomal alterations, of single sperm selected by high-magnification microscopy. To achieve this aim, we used array comparative genomic hybridization (acgh), a new method able to evaluate numerical and structural alterations of all 23,X/Y sperm chromosomes (Patassini et al., 2013). This analysis was performed on 360 single sperm with LV, small (SV) or no nuclear vacuoles obtained in semen samples from infertile patients with oligozoospermia due to testicular impairment and from normozoospermic, proven fertile men. Materials and Methods Patients and controls This study was approved by the Institutional Ethics Committee of the Hospital of Padova, Italy, Protocol number 2401P. Three patients, oligozoospermic due to testicular impairment, attending our Andrology Unit between September and November 2014 were included in the study. Three agematched proven fertile volunteers with normal sperm parameters served as controls for sperm parameters, sex hormones, testicular volumes and acgh analysis. Inclusion criteria of patients were: age years, at least 2 years of infertility, sperm count below 10 million observed at two different semen analyses performed at least 3 months apart according to World

3 Molecular karyotype of single sperm and nuclear vacuoles 2495 Health Organization guidelines (WHO, 2010), reduced testicular volumes at testicular color-doppler ultrasound and plasma FSH levels higher than 8 IU/l. All criteria had to be fulfilled in patients for inclusion. Exclusion criteria were altered karyotype, Y chromosome microdeletions, cystic fibrosis transmembrane conductance regulator gene mutations and confounding factors such as presence of sperm infections, cigarette smoking, varicocele and obesity. Inclusion criteria of controls were: age years, fatherhood during the last year and normal sperm parameters. The study flow is shown in Fig. 1. Semen samples of severe oligozoospermic patients and normozoospermic fertile controls were analyzed by high-magnification microscopy. From semen samples of patients, 68 sperm with LVs, 68 sperm with SVs and 67 without vacuoles (NVs), were retrieved individually by a micromanipulator system. Analogously, from semen samples of controls, for a total of 404 sperm. Each cell was further analyzed by whole genome amplification (WGA) and acgh to obtain its molecular karyotype. Semen sample collection and preparation Human semen samples were obtained by masturbation in sterile containers after 2 5 days of sexual abstinence. Samples were allowed to liquefy for 30 min and were examined for seminal parameters according to the World Health Organization criteria (WHO, 2010). All samples had normal viscosity, ph and semen volume. Sperm infections were excluded by sperm culture. Whole samples were washed twice by centrifugation at 300 g for 10 min in sterile phosphate-buffered saline (PBS) and a 1 ml pellet was incubated in 10-ml microdrops of polyvinylpyrrolidone solution 7% (Origio Medicult Media, Måløv, Denmark) in a Petri dish, covered with liquid paraffin (Origio Medicult Media) at room temperature for 15 min and immediately used for further analyses. Analysis of sperm at high magnification The total calculated magnification for motile sperm organelle morphology evaluation (MSOME) analysis was obtained with an Uplan Apo 100/1.40 objective lens and a 0.52 numerical aperture condenser lens. The images were captured by a DS-5M-L1 digital color video camera (Nikon) with a 1.7-cm aperture containing effective picture elements (pixels) for high-quality image production, and a color video monitor (Hansol AMW-M196A liquid crystal display). The morphologic assessment was conducted on the monitor screen, which, under the above configuration, reached a real magnification of , as determined bya 0.01-mm glass micrometer, as previously described (Garolla et al., 2008). The criteria for the evaluation of the MSOME analysis were defined according to Bartoov et al. (2002), observing sperm at higher magnification. The analysis took 30 min per patient. According to MSOME criteria, we identified three categories of sperm cells: with normal morphology and: (i) no nuclear vacuoles (NVs), (ii) at least one SV (,4% of total nuclear area) and (iii) at least one LV (.4% of total nuclear area). A total of 404 single sperm, selected one by one with a micromanipulator system (Nikon Eclipse TE2000-U equipped with Nomarski optics, HMC MC2 620/0.40 objective lens), were individually placed into a 0.2 ml PCR tube with 2 ml PBS for further acgh analysis. Only cells showing satisfactory parameters of amplification and hybridization (see below) were considered, up to 20 of each type from each subject. The final number of cells included in the study was 360. Figure 1. Study design. Semen samples of three oligozoospermic patients and three normozoospermic fertile controls were analyzed by highmagnification microscopy. From semen samples of patients and controls, 60 sperm with LVs, 60 with SVs and 60 without vacuoles (NVs) were retrieved individually by a micromanipulator system (20 of each type from each subject) giving a total of 360 sperm. Each sperm was further analyzed by WGA and acgh to obtain its molecular karyotype.

4 2496 Garolla et al. Lysis and extraction of sperm DNA and sperm decondensation This procedure was performed according to the previous study of Patassini et al. (2013). Briefly, the DNA from single sperm was extracted and amplified by the SurePlex DNA Amplification System (SurePlex WGA, BlueGnome, Cambridge, UK). The protocol was formed by a lysis phase and two rounds of PCR amplification for WGA SurePlex using a WGA procedure based on three steps: extraction and random fragmentation of genomic DNA, a pre-amplification reaction and a subsequent amplification reaction by PCR performed with flanking universal priming sites. After centrifugation, cell extraction buffer was added to each sample. Then samples were incubated in a PCR thermocycler at 758C for 10 min and at 958C for 4 min. In the second step, SurePlex Pre-Amplification mix was added to each 10 ml of sample. Decondensation was assessed with proteinase K (700 nm) and dithiothreitol (1 mm). WGA and acgh After the decondensation, in order to improve the sensitivity and the accuracy of the amplification, the last PCR-based cycling step in SurePlex has been developed in real-time, adding SYBR Green at the final concentration of Data analysis was performed by subtracting the baseline fluorescence levels at the end of each cycle, and the difference in fluorescence was plotted against the cycle number. This analysis enabled us to define a threshold of 10 cycles above which subsequent hybridization fails. The acgh procedure was divided into four steps: labeling, combination and centrifugal evaporation, hybridization and washing. In the first step, equal amounts of sperm DNA and reference DNA (SureRef. Reference male DNA) were differentially labeled with Cyanine 3 and Cyanine 5. These samples were denatured in a thermalcycler for 5 min at 948C and transferred immediately to ice for 5 min. Then, 1 ml of Klenow enzyme was added to each PCR tube to proceed with the labeling reaction. Subsequently, labeled sperm DNA and control DNA were combined, then co-precipitated adding 25 ml of COT Human DNA and concentrating by a centrifugal evaporator for 1 h at 608C, until around 3 ml remained in each tube. In the third step, labeled DNA was resuspended in dextran sulfate hybridization buffer, denatured at 758C for 10 min and hybridized under cover slips to 24sure BACarrays (BlueGnome). Then 24sure slides were placed in prepared hybridization chamber (slide box, tissue saturated with 6 ml 26 SSC/50% formamide) and incubated in a non-circulating lidded water bath for h at 47 mc. In the last step, hybridized 24sure slides were washed to remove un-hybridized DNA. The cover slip was removed from each slide by manually agitating in 26 SSC/0.05% Tween 20 at room temperature. Afterwards each slide was washed according these conditions: first wash (on stirrer) in buffer 26 SSC/0.05% Tween 20 at room temperature for 10 min; second wash (on stirrer) in buffer 16 SSC at room temperature for 10 min; third wash in buffer 0.16 SSC at 60 mc for 5 min; fourth wash (on stirrer) in buffer 0.16 SSC at room temperature for 1 min. Finally, each slide was dried by centrifugation at 170 g for 6 min. A laser scanner (G2565CA microarray scanner, Agilent, USA) was used to read the resulting images of the hybridization. Scanned images were then analyzed and quantified by BlueFuse Multi Software (Blue- Gnome). The difference in fluorescence between sample DNA and reference DNA was plotted against all chromosomes and signals above or below the fluorescence thresholds indicate, respectively, gains and losses of the DNA content. We considered for this analysis only the clones characterized by a low signal of background noise, an hybridization value of , a percentage of included clones of at least 90%, a Mean Spot Amplitude Ch1/Ch (hybridization index) and the SBR Ch1/Ch2.4 (index of the background noise). Statistical analysis All results are expressed as the mean value + SD, and categorical variables are expressed as a percentage. Comparison between groups was performed by Student s t test for continuous data after acceptance of normality with the Kolmogorov Smirnov test and by the x 2 test for categorical data. P-values of,0.05 were considered to be statistically significant. Data analysis was performed using the Statistical Package for the Social Sciences version 13.0 (SPSS, Inc., Chicago, IL, USA). Results Table I shows clinical characteristics of the three patients with testiculopathy and three age-matched proven fertile normozoospermic subjects. Patients had significantly lower sperm number, motility and morphology, higher plasma FSH and LH levels and reduced right and left testicular volume (all P, 0.01 versus controls). No significant difference in plasma testosterone levels was found in patients compared with controls. The incidence of sperm showing LV, SV and NV in patients and controls was 39.7, 57.1, 3.2% and 22.1, 69.4, 8.5%, respectively (data not shown). Figure 2 shows some examples of molecular karyotypes obtained by acgh from sperm with LV, SV or no vacuole (NV) selected by highmagnification microscopy. The LV sperm has a 23/X complex molecular karyotype (upper). The SV sperm has a 23/Y karyotype with loss of whole chromosome 14 (medium) and the NV sperm has a 23/X normal karyotype (lower). Results of array CGH are reported in Table II. Considering sperm from patients with oligozoospermia as a whole, they showed a higher mean percentage of sperm with abnormal karyotype than controls (19.4 versus 7.7%, respectively, P, 0.001), independently from the presence or absence of nuclear vacuoles. In particular, infertile patients had 38.3 and 20.0% of abnormal karyotype in sperm with LV and SV, respectively. Control subjects, had 18.3 and 5.0% abnormal karyotype in LV and SV sperm, respectively. No abnormal karyotype was found in NV sperm of both patients and controls. Comparing abnormal karyotype in oligospermic and normozoospermic patients for each group of vacuoles, statistical analysis showed significant differences between LV and NV, and between SV and NV sperm in oligozoospermic patients (P, and P, 0.01, respectively) and between LV and NV sperm in fertile controls (P, 0.01). Table III shows the type and number of alterations observed by molecular karyotype in LV, SVand NV sperm from infertile patients and fertile controls. In oligozoospermic patients, the acgh showed that among the 35 sperm with abnormal karyotype, 9.4% had partial loss or gain of chromosomes, 4.4% had XY or XX disomy, 2.8% had loss or gain of single entire chromosomes, 2.2% had complex alterations involving more chromosomes and 0.6% had sex chromosome nullisomy. In control subjects, the analysis showed 14 abnormal sperm with the following alterations: 3.8% had partial loss or gain of chromosomes, 2.2% had XY or XX disomy, 1.1% had loss or gain of single entire chromosomes and 0.6% had sex chromosome nullisomy. Interestingly, all sperm alterations were more represented in infertile patients and no complex abnormality was found in control subjects, evenwhenlv sperm were analyzed. The mostfrequentabnormality observed in both groups was the partial loss or gain of chromosomes (24/360 sperm, respectively, 9.4 and 3.8% in patients and controls). Discussion Over the last few years, it has been found that the injection of selected, morphologically normal spermatozoa into the oocyte is associated with

5 Molecular karyotype of single sperm and nuclear vacuoles 2497 Table I Clinical parameters of oligozoospermic infertile patients and normozoospermic fertile controls. Age Sperm Sperm Progressive Sperm FSH LH T Right Left (years) concentration count motility (%) morphology (U/l) (U/l) (nmol/l) testicular testicular (million/ml) (million) (%) volume volume (cc) (cc)... Oligozoospermic infertile patients Mean * 4.6* 13.6* 5.3* 13.3* 7.9* * 8.3* SD Normozoospermic fertile controls Mean SD Comparison between groups was performed by Student s t test for continuous data and by the x 2 test for categorical data. T, testosterone. *P, 0.01 versus controls. Figure 2. Examples of molecular karyotype obtained by acgh in sperm with LV, SV, or no vacuoles selected individually at high-magnification microscopy ( ) from semen samples of patients with oligozoospermia. Panel LV: sperm with LVs (left) and a 23,X,21,+8,+12,+13,+14,+16,+17,+21 complex molecular karyotype (right); panel SV: sperm with SVs (left) and a 23,Y karyotype with loss of whole chromosome 14 (right); panel NV: sperm with no vacuoles (left) with a 23,X normal molecular karyotype (right). Final magnification used for the images shown here was

6 2498 Garolla et al. higher blastocyst implantation and ongoing pregnancy rate and/or lower miscarriage rate (Bartoov et al., 2001, 2002; Berkovitz et al., 2005, 2006; Antinori et al., 2008; Vanderzwalmen et al., 2008; Balaban et al., 2011; Knez et al., 2011; Klement et al., 2013). Other studies have supported similar findings and showed that IMSI improves fertilization and blastocyst formation rates (Cassuto et al., 2009; Knez et al., 2011). Moreover, in a previous study from our group, we showed that sperm cells containing head vacuoles had a higher incidence of aneuploidies, mitochondrial impairments and DNA fragmentation (Garolla et al., 2008). The study of sperm chromosomes seems to be particularly important in those subjects with testicular damage and in particular in those patients with constitutional chromosome alterations, such as in Klinefelter syndrome, and subjects with translocations (Foresta et al., 2005; Patassini et al., Table II Results of acgh in single sperm with LVs, SVs and no vacuoles from oligozoospermic infertile patients and fertile normozoospermic controls. Sperm with abnormal karyotype... n %... Oligozoospermic infertile patients LV 23/ * SV 12/ ** NV 0/60 0 Total 35/ # Normozoospermic fertile controls LV 11/ ** SV 3/ NV 0/60 0 Total 14/ Comparison between groups was performed by the x 2 test for categorical data. LV, large vacuoles; SV, small vacuoles; NV, no vacuoles. *P, versus NV of the same group. **P, 0.01 versus NV of the same group. # P, versus fertile controls. 2013). In fact, the high aneuploidy rate frequently reported in these subjects (above 35%) compared with controls (below 8%) (Patassini et al., 2013), raises important concerns regarding the potential role in ICSI failure and the transmission of genetic diseases (In t Veld et al., 1995a,b; Liebaers et al., 1995; Loft et al., 1999; Foresta et al., 2005). The sperm vacuole is a concavity extending from the surface of the head to the nucleus through the acrosome, but its nature and significance remain still unclear. In the 1950s, by transmission electron microscopy of an ultrathin cross-section of the sperm head, it was shown that the human sperm nucleus frequently contains one or more vacuoles at different locations (Schnall, 1952; Schultz-Larsen, 1958). More recently, sperm vacuoles have been suggested to originate from spermatogenesis impairment or abnormal maturation during male genital tract transit or acrosome modification during the acrosome reaction (Perdrix et al., 2011). Most researchers suggested that nuclear vacuoles should not be considered as degenerative structures, but instead as normal features of the sperm head with no pathological significance (Fawcett, 1958; Chrzanowski, 1966; Pedersen, 1969). In contrast, some authors suggested that vacuoles are not just a polymorphism, but pose a risk for sperm abnormality at chromosome level (Bartoov et al., 2001; Vanderzwalmen et al., 2008). This hypothesis was strengthened by the demonstration that the presence of nuclear vacuoles is related to chromosome position and to chromatin integrity (Claussen et al., 2005; Finch et al., 2008; Perdrix et al., 2013). The acgh analysis involves the screening of the entire chromosome complement by DNA microarray and was used in this study to evaluate the molecular karyotype of single sperm selected by high-magnification microscopy. By this procedure, we analyzed the molecular karyotype at higher resolution than a traditional karyotype, overcoming the diagnostic limit of fluorescence in situ hybridization on sperm. The development and clinicalapplication ofacgh in recent years has revolutionized the diagnostic process in several diseases, and acgh is gradually joining or replacing standard cytogenetic techniques in a growing number of genetic and molecular laboratories (Vissers et al., 2003; Fiorentino et al., 2011). Data from the present study showed that sperm karyotype is dependent on the presence and size of nuclear vacuoles. Moreover, infertile patients with non-obstructive oligozoospermia had a higher percentage of chromosomal alterations than controls. Finally, sperm with a normal Table III Type and number of alterations at acgh analysis observed in LV, SV and NV sperm singularly retrieved from semen of oligozoospermic infertile patients and fertile normozoospermic controls. XY or XX Sex chromosome Loss/gain of single entire Loss/gain of part of Complex abnormal disomy, n (%) nullisomy, n (%) chromosomes, n (%) chromosomes, n (%) karyotype, n (%)... Oligozoospermic infertile patients LV (n ¼ 60) 5 (8.3) 1 (1.7) 3 (5.0) 10 (16.6) 4 (6.7) SV (n ¼ 60) 3 (5.0) 0 2 (3.3) 7 (11.7) 0 NV (n ¼ 60) Total (4.4) 1 (0.6) 5 (2.8) 17 (9.4) 4 (2.2) Normozoospermic fertile controls LV (n ¼ 60) 3 (5.0) 1 (1.7) 2 (3.3) 5 (8.3) 0 SV (n ¼ 60) 1 (1.7) (3.3) 0 NV (n ¼ 60) Total (2.2) 1 (0.6) 2 (1.1) 7 (3.8) 0

7 Molecular karyotype of single sperm and nuclear vacuoles 2499 morphology and no vacuole always showed a normal molecular karyotype both in patients and controls. It is worthy of mention that just some of all sperm retrieved from patients had an abnormal karyotype (19.4%). However, we have to consider that chromosomal alterations represent only a mirror of more subtle sperm DNA alterations, such as chromatin mispackaging and DNA fragmentation. Future application of acgh on sperm might give important information on the biology and pathophysiology of spermatogenesis and sperm chromosome aberrations in healthy fertile subjects, as well as in patients at higher risk of producing unbalanced sperm, such as infertile men, carriers of karyotype anomalies, men with advanced age, subjects treated with chemotherapy and partners of couples with repeated miscarriage and repeated failure of assisted reproduction techniques. In these patients, pre-implantation genetic screening and PGD could also be considered to reduce the impact of paternal abnormalities on embryo aneuploidy. In conclusion, our data demonstrate that molecular karyotypes of sperm from infertile patients show a higher incidence of abnormalities and these alterations are related to the presence of nuclear vacuoles. Further and larger studies performed in patients with severe testicular failure are needed to understand if sperm selection for ICSI by high-magnification microscopy could represent a reliable method to improve the ICSI outcome and to reduce the transmission of genes abnormalities to offspring. Authors roles A.G. study design, data interpretation and writing text; B.S. data collection and writing text; I.C. semen analysis, data collection, writing text; D.P. enrolling subjects and statistical analysis; M.G. enrolling subjects and statistical analysis; A.B. generation of tables and figures; M.M. semen and molecular analyses; E.S. molecular analyses; A.F. study design and data interpretation; C.F. study design and data interpretation. Funding No external funding was sought for this study. Conflict of interest All authors have no conflict of interest to declare regarding this manuscript. References Antinori M, Licata E, Dani G, Cerusico F, Versaci C, D Angelo D, Antinori S. Intracytoplasmic morphologically selected sperm injection: a prospective randomized trial. Reprod Biomed Online 2008;16: Balaban B, Yakin K, Alatas C, Oktem O, Isiklar A, Urman B. Clinical outcome of intracytoplasmic injection of spermatozoa morphologically selected under high magnification: a prospective randomized study. Reprod Biomed Online 2011;22: Bartoov B, Berkovitz A, Eltes F. Selection of spermatozoa with normal nuclei to improve the pregnancy rate with intracytoplasmic sperm injection. N Engl J Med 2001;345: Bartoov B, Berkovitz A, Eltes F, Kogosowski A, Menezo Y, Barak Y. Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome. J Androl 2002;23:1 8. Berkovitz A, Eltes F, Yaari S, Katz N, Barr I, Fishman A, Bartoov B. The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Hum Reprod 2005;20: Berkovitz A, Eltes F, Ellenbogen A, Peer S, Feldberg D, Bartoov B. Does the presence of nuclear vacuoles in human sperm selected for ICSI affect pregnancy outcome? Hum Reprod 2006;21: Boitrelle F, Ferfouri F, Petit JM, Segretain D, Tourain C, Bergere M, Bailly M, Vialard F, Albert M, Selva J. Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of chromatin condensation. Hum Reprod 2011; 26: Bonduelle M, Van Assche E, Joris H, Keymolen K, Devroey P, Van Steirteghem A, Liebaers I. Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Hum Reprod 2002;17: Cassuto NG, Bouret D, Plouchart JM, Jellad S, Vanderzwalmen P, Balet R, Larue L, Barak Y. A new real-time morphology classification for human spermatozoa: a link for fertilization and improved embryo quality. Fertil Steril 2009;92: Chrzanowski S. Ultrastructure of human spermatozoon. Pol Med J 1966; 5: Claussen U. Chromosomics. Cytogenet Genome Res 2005;111: Erenpreiss J, Spano M, Erenpreisa J, Bungum M, Giwercman A. Sperm chromatin structure and male fertility: biological and clinical aspects. Asian J Androl 2006;8: Fawcett DW. The structure of the mammalian spermatozoon. Int Rev Cytol 1958;7: Figueira Rde C, Braga DP, Setti AS, Iaconelli A Jr, Borges E Jr. Morphological nuclear integrity of sperm cells is associated with preimplantation genetic aneuploidy screening cycle outcomes. Fertil Steril 2011;95: Finch KA, Fonseka KG, Abogrein A, Ioannou D, Handyside AH, Thornhill AR, Hickson N, Griffin DK. Nuclear organization in human sperm: preliminary evidence for altered sex chromosome centromere position in infertile males. Hum Reprod 2008;23: Fiorentino F, Spizzichino L, Bono S, Biricik A, Kokkali G, Rienzi L, Ubaldi FM, Iammarrone E, Gordon A, Pantos K. PGD for reciprocal and Robertsonian translocations using array comparative genomic hybridization. Hum Reprod 2011;26: Foresta C, Garolla A, Bartoloni L, Bettella A, Ferlin A. Genetic abnormalities among severely oligospermic men who are candidates for intracytoplasmic sperm injection. J Clin Endocrinol Metab 2005;90: Franco JG Jr, Baruffi RL, Mauri AL, Petersen CG, Oliveira JB, Vagnini L. Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI. Reprod Biomed Online 2008;17: Garolla A, Fortini D, Menegazzo M, De Toni L, Nicoletti V, Moretti A, Selice R, Engl B, Foresta C. High-power microscopy for selecting spermatozoa ICSI by physiological status. Reprod Biomed Online 2008; 17: In t Veld P, Brandenburg H, Verhoeff A, Dhont M, Los F. Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet 1995a; 16: In t Veld PA, van Opstal D, Van den Berg C, Van Ooijen M, Brandenburg H, Pijpers L, Jahoda MG, Stijnen TH, Los FJ. Increased incidence of cytogenetic abnormalities in chorionic villus samples from pregnancies established by in vitro fertilization and embryo transfer (IVF-ET). Prenat Diagn 1995b; 15: Kacem O, Sifer C, Barraud-Lange V, Ducot B, De Ziegler D, Poirot C, Wolf J. Sperm nuclear vacuoles, as assessed by motile sperm organellar morphological examination, are mostly of acrosomal origin. Reprod Biomed Online 2010;20: Klement AH, Koren-Morag N, Itsykson P, Berkovitz A. Intracytoplasmic morphologically selected sperm injection versus intracytoplasmic sperm

8 2500 Garolla et al. injection: a step toward a clinical algorithm. Fertil Steril 2013;99: Knez K, Zorn B, Tomazevic T, Vrtacnik-Bokal E, Virant-Klun I. The IMSI procedure improves poor embryo development in the same infertile couples with poor semen quality: a comparative prospective randomized study. Reprod Biol Endocrinol 2011;9:123. Krausz C. Male infertility: pathogenesis and clinical diagnosis. Best Pract Res Clin Endocrinol Metab 2011;25: Liebaers I, Bonduelle M, Van Assche E, Devroey P, Van Steirteghem A. Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet 1995;346:1095. Loft A, Petersen K, Erb K, Mikkelsen AL, Grinsted J, Hald F, Hindkjaer J, Nielsen KM, Lundstrom P, Gabrielsen A et al. A Danish cohort of 730 infants born after intracytoplasmic sperm injection (ICSI) Hum Reprod 1999;14: Montjean D, Belloc S, Benkhalifa M, Dalleac A, Menezo Y. Sperm vacuoles are linked to capacitation and acrosomal status. Hum Reprod 2012;27: Pang MG, Hoegerman SF, Cuticchia AJ, Moon SY, Doncel GF, Acosta AA, Kearns WG. Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum Reprod 1999;14: Patassini C, Garolla A, Bottacin A, Menegazzo M, Speltra E, Foresta C, Ferlin A. Molecular karyotyping of human single sperm by arraycomparative genomic hybridization. PLoS One 2013;8:e Pedersen H. Ultrastructure of the ejaculated human sperm. Z Zellforsch 1969; 94: Perdrix A, Rives N. Motile sperm organelle morphology examination (MSOME) and sperm head vacuoles: state of the art in Hum Reprod Update 2013;19: Perdrix A, Travers A, Chelli MH, Escalier D, Do Rego JL, Milazzo JP, Mousset-Simèon N, Macè B, Rives N. Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles. Hum Reprod 2011;26: Perdrix A, Travers A, Clatot F, Sibert L, Mitchell V, Jumeau F, Macé B, Rives N. Modification of chromosomal architecture in human spermatozoa with large vacuoles. Andrology 2013;1: Schnall MD. Electron microscopic study of human spermatozoa. Fertil Steril 1952;3: Schultz-Larsen J. The morphology of the human sperm; electron microscopic investigations of the ultrastructure. Acta Path Microbiol Scand Suppl 1958; 44: TeixeiraDM, BarbosaMA, FerrianiRA, Navarro PA, Raine-FenningN, NastriCO, Martins WP. Regular (ICSI) versus ultra-high magnification (IMSI) sperm selection for assisted reproduction. Cochrane Database Syst Rev 2013; 25:7. Vanderzwalmen P, Hiemer A, Rubner P, Bach M, Neyer A, Stecher A, Uher P, Zintz M, Lejeune B, Vanderzwalmen S et al. Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles. Reprod BioMed Online 2008;17: Vissers LE, de Vries BB, Osoegawa K, Janssen IM, Feuth T, Choy CO, Straatman H, van der Vliet W, Huys EH, van Rijk A et al. Array-based comparative genomic hybridization for the genomewide detection of submicroscopic chromosomal abnormalities. Am J Hum Genet 2003; 73: Ward WS. Function of sperm chromatin structural elements in fertilization and development. Mol Hum Reprod 2010;16: Watanabe S, Tanaka A, Fujii S, Mizunuma H. No relationship between chromosome aberrations and vacuole-like structures on human sperm head. Hum Reprod 2009;24: World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. Cambridge, UK: Cambridge University Press, Zamboni L. The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality. Fertil Steril 1987;48: