Is intracytoplasmic morphologically selected sperm injection (IMSI) beneficial in the first ART cycle? A multicentric randomized controlled trial

Transcription

1 ISSN: ORIGINAL ARTICLE Correspondence: Jean Parinaud, Medecine de la Reproduction, CHU de Toulouse, Toulouse, France. Keywords: indications, intracytoplasmic morphologically selected sperm injection, intracytoplasmic sperm injection, male infertility Received: 29-Mar-2013 Revised: 15-May-2013 Accepted: 16-May-2013 doi: /j x Is intracytoplasmic morphologically selected sperm injection (IMSI) beneficial in the first ART cycle? A multicentric randomized controlled trial 1,2 R. D. Leandri, 1,2 A. Gachet, 3 J. Pfeffer, 4 C. Celebi, 5 N. Rives, 6 F. Carre-Pigeon, 7 O. Kulski, 8 V. Mitchell and 1,2 J. Parinaud 1 Medecine de la Reproduction, CHU de Toulouse, Toulouse, France, 2 EA 3694, Groupe de Recherche en Fertilite Humaine, Universite Paul Sabatier, Toulouse, France, 3 Laboratoire ZTP, Clinique de la DHUYS, Bagnolet, France, 4 Laboratoire de Biologie de la Reproduction, CMCO Schiltigheim, CHRU de Lille, Schiltigheim, France, 5 Laboratoire de Biologie de la Reproduction-CECOS, EA 4308 «Gametogenese et Qualite du Gamete», CHU H^opitaux de Rouen, Rouen, France, 6 Laboratoire de Biologie de la Reproduction-CECOS, CHU de Reims, Reims, France, 7 Laboratoire de Biologie de la Reproduction, H^opital des 4 Villes, Sevres, France, and 8 EA4308-Institut de la Biologie de la Reproduction, CHRU de Lille, Lille, France SUMMARY Intracytoplasmic morphologically selected sperm injection (IMSI), by selecting spermatozoa at high magnification improves the outcome of intracytoplasmic sperm injection (ICSI) mainly after several failures. However, only few monocentric randomized studies are available and they do not analyse results as a function of sperm characteristics. In 255 couples attempting their first assisted reproductive technology (ART) attempt for male infertility (motile sperm count < after sperm selection, but at least spermatozoa per ejaculate to allow a detailed analysis of sperm characteristics), a prospective randomized trial was performed to compare the clinical outcomes of IMSI and ICSI and to evaluate the influence of sperm characteristics on these outcomes. IMSI did not provide any significant improvement in the clinical outcomes compared with ICSI neither for implantation (24% vs. 23%), nor clinical pregnancy (31% vs. 33%) nor live birth rates (27% vs. 30%). Moreover, the results of IMSI were similar to the ICSI ones whatever the degree of sperm DNA fragmentation, nuclear immaturity and sperm morphology. These results show that IMSI instead of ICSI has no advantage in the first ART attempts. However, this does not rule out IMSI completely and more randomized trials must be performed especially regarding patients carrying severe teratozoospermia, or high sperm DNA fragmentation levels or having previous ICSI failures. INTRODUCTION The development of intracytoplasmic sperm injection (ICSI) marked an important advance in the field of assisted reproduction. However, one remaining problem is the selection step of the spermatozoa for microinjection. The optics used for visualization of spermatozoa do not provide a sufficient resolution to allow identification of small defects and morphological abnormalities whereas the morphological quality of spermatozoa plays a major role in fertilization, implantation and pregnancy (De Vos et al., 2003). By increasing the resolution of the optics ( ), intracytoplasmic morphologically selected sperm injection (IMSI) is able to select motile spermatozoa devoided of any structural defects and to optimize the outcome of ICSI (Bartoov et al., 2003). Several studies have demonstrated that IMSI significantly improves embryo quality (Bartoov et al., 2003; Berkovitz et al., 2006b) implantation and pregnancy rates (Berkovitz et al., 2006a). The presence of nuclear vacuoles in the injected spermatozoa was reported to be the most relevant parameter that might significantly impact the clinical results of ICSI (Berkovitz et al., 2006a). Vanderzwalmen et al. (2008) showed that, in 25 patients where sibling oocytes were injected with spermatozoa graded according to the extent and size of nuclear vacuoles, the blastocyst formation and quality were closely associated with the grade of the spermatozoa used. No blastocysts were obtained when spermatozoa with large vacuoles were used. These data suggested that the most predictive factor of sperm quality was the size of the sperm head vacuoles 692 Andrology, 2013, 1, American Society of Andrology and European Academy of Andrology

2 IMSI VS. ICSI FOR THE FIRST ATTEMPTS (Vanderzwalmen et al., 2008). However, the origin of these vacuoles is still debated. Most of publications support a nuclear origin. Some authors link the presence of large vacuoles with an increase in fragmented DNA (Franco et al., 2008; Oliveira et al., 2010), whereas other data are in agreement with chromatin condensation defects (Boitrelle et al., 2011; Perdrix et al., 2011; Franco et al., 2012). On the other hand, an acrosomic origin of these vacuoles has also been suggested (Kacem et al., 2010; Montjean et al., 2012). However, the first data about IMSI came from non-randomized trials with small and selected populations (Bartoov et al., 2001, 2002, 2003; Berkovitz et al., 2005; Hazout et al., 2006). The first prospective randomized study was published by Antinori and co-workers, which reported higher pregnancy and implantation rates in severe male factor infertility cases following IMSI. IMSI resulted in significantly higher implantation (17.3% vs. 11.3%; p = 0.007) and clinical pregnancy rates (39.2 vs. 26.5%, p = 0.004) compared with conventional ICSI. The clinical benefit was more significant in patients with two or more previously failed treatment attempts. However, no precision was addressed on the quality of the spermatozoa used (Antinori et al., 2008). More recently, a randomized trial including 168 couples attempting their first assisted reproductive technology (ART), showed that IMSI did not provide a significant improvement in the clinical outcome compared with ICSI although a trend for higher implantation (28.9% vs. 19.5%), clinical pregnancy (54.0% vs. 44.4%) and live birth rates (43.7% vs. 38.3%) was observed in the IMSI group. However, in case of severe male factor patients, IMSI resulted in a significantly higher implantation rate (29.6% vs. 15.2%), p = 0.01) (Balaban et al., 2011). Using a multicentre randomized trial, this study aimed to determine firstly if IMSI is beneficial in the first ART cycle compared with ICSI, and secondly to investigate the indications of IMSI according to sperm characteristics. MATERIALS AND METHODS Patients This prospective randomized study included couples attending their first ART attempt, in seven ART centres in France (H^opital Paule de Viguier CHU Toulouse, CMCO-Schiltigheim, H^opital Jeanne de Flandre-Lille, CHU-H^opitaux de Rouen, CH des 4 Villes-Sevres-Saint Cloud-Chaville-Ville d Avray, Laboratoire ZTP-Clinique de la DHUYS, Bagnolet, H^opital Maison Blanche-Reims) from September 2008 to December The calculation of the number of patients leads to a total population of 458 couples (229 per harm) to identify a difference of 10 points in live birth rates between IMSI and ICSI (from 25 to 35%), with 80% power and 5% error risk. However, as the evaluation at mid-inclusion could not evidence any difference between the two techniques, the trial was stopped prematurely. A total of 255 couples, undergoing their first ICSI attempt, entered the study. The inclusion criteria were as follows: indications of ICSI for male infertility with use of fresh ejaculated spermatozoa with at least 3 millions of spermatozoa in the ejaculate and less than 1 million of motile spermatozoa recovered after density gradient with whatever sperm morphology. We excluded patients with female partners older than 39 years and/or with day 3 follicle stimulating hormone (FSH) level over 9 UI/L. In each centre, once the patient informed consent was obtained, the couples were randomly allocated the day of oocyte retrieval to receive IMSI or ICSI using computer generated random numbers (unrestricted randomization list). All couples were blinded regarding their treatment assignment for the duration of the study. The following groups were formed: group 1 was named ICSI group and included 139 patients who would receive a conventional ICSI procedure; group 2 was named IMSI group and included 116 patients who would receive an IMSI procedure in the same period. In each group, only one cycle was performed per couple. The study was approved by the Comite de Protection des Personnes SOOM II (no ) and by Agence Francßaise de Securite Sanitaire des Produits de Sante (no. B ). Semen preparation Semen samples were collected by masturbation, directly in each of the seven centres after 3 5 days of sexual abstinence and were processed for analysis after liquefaction for 20 min at 37 C. Semen characteristics [volume, sperm concentration (10 6 /ml), progressive motility (WHO grade a b combined:%), vitality (%)] were interpreted considering references provides in WHO guidelines (World Health Organization, 2010). A part of the spermatozoa was frozen for further analysis on morphology and quality of sperm nucleus and the frozen specimens centralized in the ART centre of Toulouse to minimize inter-observers variations. Sperm morphology Sperm morphology was evaluated using a computer-assisted sperm analysis on slides of fresh semen stained with the Kit RAL 555 (RAL Diagnostics, Martillac, France) with the Hamilton Thorn Version 12.3 software according to Kruger s classification (Kruger et al., 1995; Marnet et al., 2000). Sperm morphology was also centralized in the ART centre of Toulouse. Aniline blue (AB) staining After thawing, a 5-lL aliquot of cryopreserved spermatozoa was spread on a glass slide and left to dry. The smears were then fixed in 3% buffered glutaraldehyde in phosphate buffer saline (PBS) for 30 min. Slides were then stained with 5% aqueous AB mixed with 4% acetic acid (ph 3.5) for 5 min and washed twice with distilled water (Auger et al., 1990). A total of 100 spermatozoa per slide were evaluated. The threshold of nuclear immaturity is 20% (Hammadeh et al., 1998). DNA fragmentation assay DNA fragmentation was measured with the TUNEL assay with the in situ Cell Death Detection Kit Fluorescein, according to manufacturer s protocol (Roche Diagnostics, Indianapolis, IN), with slight modifications. From frozen semen, the pellet of spermatozoa was fixed in a solution of methanol : acetic acid (3 : 1) for 30 min at 4 C, then, spread on slides and dried at room temperature for 4 h. Slides were rinsed twice in PBS before being permeabilized with 0.1% Triton X100 in 0.1% sodium citrate for 2 min at 4 C. Slides were then incubated in a humidified chamber at 37 C for 60 min with terminal deoxyribonucleotidyl transferase (TdT) that mediates dutp nick-end labelling to allow DNA elongation American Society of Andrology and European Academy of Andrology Andrology, 2013, 1,

3 R. D. Leandri et al. Slides were rinsed twice in PBS and counterstained with 1 mg/ml 4,6 diamidoino-2-phenylindole. Negative (omitting TdT from the reaction mixture) and positive (using only Dnase I, 1 mg/ml for 30 min at room temperature) controls were tested for each series of samples. The presence of DNA fragmentation was detected if fluorescein fluorescence (green) was present (TUNEL positive). A total of 100 spermatozoa per slide were evaluated. The DNA fragmentation index (DFI) is equal to the percentage of sperm positive total population of spermatozoa observed. IMSI procedure In all centres, an aliquot of the sperm preparation was placed in a glass-bottomed dish (WillCo-dish; WillCo Wells BV, Amsterdam, The Netherlands) and examined by Nomarski interference contrast microscopy with a Leica (Leica microsystems, Nanterre, France) DFC-280 camera mounted on a Leica DMI 6000 microscope with an immersion objective lens 9100 and camera magnification 91. The scoring system for the evaluation and selection of the injected spermatozoa was based on the classification published by Perdrix et al. (2012): Type 0: spermatozoa without vacuole. Type 1: spermatozoa with vacuoles occupying 0 5.9% of the nuclear surface. Type 2: spermatozoa with vacuoles occupying % of the nuclear surface. Type 3: spermatozoa with vacuoles occupying over than 12.4% of the nuclear surface. If available, type 0 spermatozoa were preferentially injected, then type 1 if there was no type 0, then type 2 if there was no type 1 and at last type 3 if no other spermatozoa were found. Before the first inclusion, all participants met for training to reduce inter-observer variations. Conventional ICSI procedure The sperm selection for microinjection was performed at a magnification of Spermatozoa with severe head shape defects clearly seen under this magnification (pin, amorphous, tapered, round and multinucleated head) were excluded from microinjection into the oocyte. Embryo culture Although there is no clear evidence for the superiority of a specific medium on birth rates as recently reviewed (Mantikou et al., 2013), the impact of the entire embryo culture system (combining the type of culture medium, volume of the drops, gas phases, plastic, oil, etc.) is far from known. As randomization was performed in each centre, all laboratory procedures used from oocyte retrieval to embryo transfer (except the micro-injection step) were equally applied to oocytes, zygotes and embryos from both IMSI and ICSI groups. The fertilization of the oocytes was checked the next day, h after microinjection. The zygotes were considered to be normal when they had two pronuclei and two polar bodies, and abnormal when they did not develop further (non-cleaved zygotes) or were triploid (with three pronuclei). The embryos from both ICSI and IMSI groups were transferred according to usual procedures of each centre on day 2 or 3 after oocyte retrieval as it has been shown to have no influence on live birth rates (Oatway et al., 2004). Statistical analysis Implantation rate was defined as the ratio of the number of gestational sacs with foetal heart beat to the number of transferred embryos. Statistical analysis was performed using the software StatView â (Abacus Concepts Inc., Berkeley, CA). Percentages were compared by the Chi-squared test, averages by Student s t-test. To compare the results based on the percentage of different sperm parameters, they were divided into three groups (<25th percentile, 25th 75th percentile, >75th percentile). A p value lower than 0.05 was considered as statistically significant. RESULTS The characteristics of the patients are summarized in Table 1. They were similar between IMSI and ICSI groups in term of male and female age, semen parameters and female FSH levels. Cycle characteristics (number of injected FSH units, estradiol levels, stimulation parameters, number of collected oocytes, number of mature oocytes, number of transferred embryos, etc.) did not show any statistical difference between the two groups (Table 2). No statistically significant difference was observed between the two groups for DFI, AB staining and percentage of normal forms. As shown in Table 2, no statistically significant difference was observed between the two groups with regard to the rates of implantation (IMSI: 24%; ICSI: 22%; NS), ongoing pregnancy (IMSI: 31%: ICSI: 33%, NS) and birth (IMSI: 27% ICSI: 30%; NS). However, the fertilization rate was significantly lower in the IMSI group (56 25 vs , p < 0.05), as well as the total number of embryos ( vs ; p < 0.05) and the number of frozen embryos ( vs ; p < 0.05). As we have observed differences in fertilization rates among the centres, to elucidate if the drop of fertilization could impact the clinical outcomes, we compared the clinical outcomes when Table 1 Demographic data of the population of males and females participating in the study Total ICSI IMSI Statistical comparison Number of cycles NS Female age NS Male age NS Infertility NS length (month) Female FSH (UI/L) NS Sperm parameters Volume (ml) NS Sperm NS count (10 6 /ml) Motility (%) NS Vitality (%) NS Number of motile NS spermatozoa (10 6 ) recovered after preparation % of morphologically NS normal spermatozoa DNA NS fragmentation (%) Immature nuclei (%) NS Values are expressed as mean SD. p < 0.05 is considered significant. FSH: follicle stimulating hormone; ICSI: intracytoplasmic sperm injection; IMSI: intracytoplasmic morphologically selected sperm injection; NS: not significant. 694 Andrology, 2013, 1, American Society of Andrology and European Academy of Andrology

4 IMSI VS. ICSI FOR THE FIRST ATTEMPTS Table 2 Results of ICSI and IMSI cycles Total ICSI IMSI Statistical comparison Ovarian stimulation protocol Short agonist (%) NS Long agonist (%) NS Antagonist (%) NS Total injected FSH units NS No. of collected oocytes NS No. of metaphase-ii oocytes NS Fertilization rate (%) p < 0.05 No. of obtained embryos p < 0.05 No. of transferred embryos NS No. of frozen embryos p < 0.05 Implantation rate (%) NS Clinical pregnancy rate (%) NS Delivery rate (%) NS Values are expressed as mean SD. p < 0.05 is considered significant. FSH: follicle stimulating hormone; ICSI: intracytoplasmic sperm injection; IMSI: intracytoplasmic morphologically selected sperm injection; No: number; NS: not significant. eliminating centres showing a significant decrease in fertilization rate with IMSI. In the remaining 75 IMSI cycles, implantation rate (26%) and live birth rate (33%) were not different from those observed in the 89 ICSI cycles (25 and 35% respectively). Furthermore, in the remaining centres, the mean number of frozen embryos is not significantly inferior in the IMSI group (n = ) compared with the ICSI group ( ). To detect a potential male sub-population that could benefit from IMSI, we analysed the results according to sperm quality (morphology, DNA fragmentation and nuclear maturity). No improvement was observed in clinical outcomes after IMSI whatever the sperm characteristics (Table 3). Finally, to evaluate the impact of sperm vacuoles on implantation rates, single embryo transfers (SET) were analysed (n = 42). SET were performed in all centres either when no other embryo (n = 9) was available or if the chances of pregnancy were estimated high to avoid twin pregnancies (n = 33). Although the number of cases is relatively low, no significant difference was found according to the quality of injected spermatozoa (Table 4). Table 3 Comparison of implantation rates between IMSI and ICSI according to sperm characteristics Total ICSI IMSI Statistical comparison Implantation rate according DFI (%) <10% NS 10 22% NS >22% NS Implantation rate according aniline blue staining (%) <10% NS 10 23% NS >23% NS Implantation rate according the percentage of morphologically normal spermatozoa (%) <1% NS 1 7% NS >7% NS Implantation rate (%) according the number of motile spermatozoa recovered after preparation (10 6 ) < NS NS > NS DFI: DNA fragmentation index; ICSI: intracytoplasmic sperm injection; IMSI: intracytoplasmic morphologically selected sperm injection; NS: not significant. Table 4 Implantation rates after single embryo transfer according to the morphology of injected sperm according to used classification (Perdrix et al., 2012) Implantation rate Type of spermatozoa 0 1/10 (10) NS 1 6/23 (26) 2 1/8 (13) 3 0/1 Values in parenthesis are expressed in percentage. Statistical comparison DISCUSSION The results of this study did not show real benefit for IMSI compared with the conventional ICSI method in case of the first ART attempt, the live birth rate being similar with both techniques (30% in ICSI vs. 27% in IMSI). Surprisingly, we found no impact of vacuoles on implantation rates in the 42 cases of SET, in contradiction of Vanderzwalmen s study, which reported low blastocyst development when injecting spermatozoa with large vacuoles (Vanderzwalmen et al., 2008). However, it must be noticed that in our study, most of the injected spermatozoa had few vacuoles (type 0 or 1 according to Perdrix classification; Perdrix et al., 2012). It must be pointed out that the implantation rate in ICSI group in this study is higher than those reported in Antinori s and Balaban s studies and similar to the rates they obtained with IMSI. Moreover, in the first randomized studies the improvement in IMSI was especially marked after at least two failures of ICSI (Antinori et al., 2008; Balaban et al., 2011; Wilding et al., 2011). Indeed, the pregnancy rate was not significantly higher in IMSI for couples attending their first attempt, although a trend in favour of IMSI was observed (Antinori et al., 2008; Balaban et al., 2011). Therefore, it could be postulated that IMSI could have a beneficial effect only in poor prognosis patients. It must be noticed that the fertilization rate obtained for the 116 attempts of IMSI is significantly lower than that of ICSI. We have no clear explanation of this observation, but one can hypothesize that the technical constraints of IMSI (time) affect the quality of gametes. Such a deleterious effect of IMSI has not been reported in other studies and the fertilization rates were found identical in ICSI and IMSI. However, it must be pointed out that in previous RCT, either the sperm parameters are clearly different from ours (mean sperm concentration around 2013 American Society of Andrology and European Academy of Andrology Andrology, 2013, 1,

5 R. D. Leandri et al. 40 millions/ml and sperm mobility around 40% in the Balaban study on first attempts, Balaban et al., 2011) or the rank of the attempt is not specified (Wilding et al., 2011). We believe that the lower fertilization rate observed after IMSI is not directly responsible for the absence of benefit of IMSI on the clinical outcomes as it has been observed even in the centres where fertilization rate is not inferior after IMSI. It is noteworthy to note that this decrease in fertilization after IMSI results in a decreased number of frozen embryos and therefore could negatively affect the cumulative live birth rate per cycle compared with ICSI, an outcome not studied here. Vacuoles have been reported to be associated with DNA fragmentation, which is known to negatively influence embryo development (Franco et al., 2008; Garolla et al., 2008). Therefore, it could be postulated that IMSI, by selecting spermatozoa with no or few vacuoles, could enhance outcome in cases with high DNA fragmentation levels (Hazout et al., 2006; Junca et al., 2009). When studying the results as a function of DFI percentage, we found no difference between ICSI and IMSI. However, the rate of DNA fragmentation in our population was relatively low and we cannot exclude a beneficial effect of IMSI in cases with high DNA fragmentation levels. As Boitrelle et al. (2011) have reported that vacuoles are related to chromatin condensation defects, IMSI could be of interest in case of high levels of AB staining. In this study, we have found no difference for implantation rates between IMSI and ICSI even when the AB stained level was high (>23%). As DNA fragmentation and chromatin condensation were assessed after freezing thawing procedures, we cannot exclude of slight change in comparison with fresh samples as reported by Boitrelle et al. (2011). However, the same procedure was applied to ICSI and IMSI samples thus lowering possible bias. Previous data have reported that morphologically abnormal spermatozoa have a negative impact on fertilization and embryo quality, even when ICSI is performed (Parinaud et al., 1993; Janny & Menezo, 1994; De Vos et al., 2003; Berkovitz et al., 2005, 2006a). Therefore, it could be postulated that IMSI, by selecting spermatozoa under magnification could improve outcomes in patients with teratozoospermia (Knez et al., 2012). When studying results as a function of percentage of normal forms, no statistical difference between ICSI and IMSI has been demonstrated. However, a trend was observed in cases with low percentage of normal forms (<1%) and therefore we cannot exclude that IMSI could have a beneficial effect in patients with severe teratozoospermia as shown by others (Knez et al., 2012). These lacks of any benefit of IMSI could be explained by the fact that in the labs used to perform IMSI, the choice of the spermatozoon to be injected is made more carefully so that those carrying large vacuoles, which can be seen by careful examination at a 4009 magnification, are not injected even with conventional ICSI. Our trial concludes to the lack of interest to replace ICSI by IMSI in the first attempt. However, this does not rule out IMSI completely and more randomized trials must be performed especially regarding patients carrying severe teratozoospermia, or high sperm DNA fragmentation levels or having previous ICSI failures. AUTHORS CONTRIBUTION AG performed sperm analyses (DNA fragmentation, aniline blue staining morphology), RL, NR, CC, FCP, OK, VM and JP recruited patients and performed ICSI and IMSI. JP designed the study. All participated to the redaction of the article. FUNDING This work was supported by a grant from the French Ministry of Health (Clinical Research Hospital Program 2008; no ). REFERENCES Antinori M, Licata E, Dani G, Cerusico F, Versaci C, d Angelo D et al. (2008) Intracytoplasmic morphologically selected sperm injection: a prospective randomized trial. Reprod Biomed Online 16, Auger J, Mesbah M, Huber C & Dadoune JP. (1990) Aniline blue staining as a marker of sperm chromatin defects associated with different semen characteristics discriminates between proven fertile and suspected infertile men. Int J Androl 13, Balaban B, Yakin K, Alatas C, Oktem O, Isiklar A & Urman B. (2011) Clinical outcome of intracytoplasmic injection of spermatozoa morphologically selected under high magnification: a prospective randomized study. Reprod Biomed Online 22, Bartoov B, Berkovitz A & Eltes F. (2001) Selection of spermatozoa with normal nuclei to improve the pregnancy rate with intracytoplasmic sperm injection. N Engl J Med 345, Bartoov B, Berkovitz A, Eltes F, Kogosowski A, Menezo Y & Barak Y. (2002) Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome. J Androl 23, 1 8. Bartoov B, Berkovitz A, Eltes F, Kogosovsky A, Yagoda A, Lederman H et al. (2003) Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertil Steril 80, Berkovitz A, Eltes F, Yaari S, Katz N, Barr I, Fishman A et al. (2005) The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Hum Reprod 20, Berkovitz A, Eltes F, Ellenbogen A, Peer S, Feldberg D & Bartoov B. (2006a) Does the presence of nuclear vacuoles in human sperm selected for ICSI affect pregnancy outcome? Hum Reprod 21, Berkovitz A, Eltes F, Lederman H, Peer S, Ellenbogen A, Feldberg B et al. (2006b) How to improve IVF-ICSI outcome by sperm selection. Reprod Biomed Online 12, Boitrelle F, Ferfouri F, Petit JM, Segretain D, Tourain C, Bergere M et al. (2011) Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of chromatin condensation. Hum Reprod 26, De Vos A, Van De Velde H, Joris H, Verheyen G, Devroey P & Van Steirteghem A. (2003) Influence of individual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection. Fertil Steril 79, Franco JG, Baruffi RL, Mauri AL, Petersen CG, Oliveira JB & Vagnini L. (2008) Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI. Reprod Biomed Online 17, Franco JG Jr, Mauri AL, Petersen CG, Massaro FC, Silva LF, Felipe V et al. (2012) Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa. Int J Androl 35, Garolla A, Fortini D, Menegazzo M, De Toni L, Nicoletti V, Moretti A et al. (2008) High-power microscopy for selecting spermatozoa for ICSI by physiological status. Reprod Biomed Online 17, Hammadeh ME, Stieber M, Haidl G & Schmidt W. (1998) Association between sperm cell chromatin condensation, morphology based on strict criteria, and fertilization, cleavage and pregnancy rates in an IVF program. Andrologia 30, Hazout A, Dumont-Hassan M, Junca AM, Cohen Bacrie P & Tesarik J. (2006) High-magnification ICSI overcomes paternal effect resistant to conventional ICSI. Reprod Biomed Online 12, Andrology, 2013, 1, American Society of Andrology and European Academy of Andrology

6 IMSI VS. ICSI FOR THE FIRST ATTEMPTS Janny L & Menezo YJ. (1994) Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol Reprod Dev 38, Junca AM, Cohen-Bacrie P, Belloc S, Dumont M & Menezo Y. (2009) Teratozoospermia at the time of intracytoplasmic morphologically selected sperm injection (IMSI). Gynecol Obstet Fertil 37, Kacem O, Sifer C, Barraud-Lange V, Ducot B, De Ziegler D, Poirot C et al. (2010) Sperm nuclear vacuoles, as assessed by motile sperm organellar morphological examination, are mostly of acrosomal origin. Reprod Biomed Online 20, Knez K, Tomazevic T, Zorn B, Vrtacnik-Bokal E & Virant-Klun I. (2012) Intracytoplasmic morphologically selected sperm injection improves development and quality of preimplantation embryos in teratozoospermia patients. Reprod Biomed Online 25, Kruger TF, du Toit TC, Franken DR, Menkveld R & Lombard CJ. (1995) Sperm morphology: assessing the agreement between the manual method (strict criteria) and the sperm morphology analyzer IVOS. Fertil Steril 63, Mantikou E, Youssef MA, van Wely M, van der Veen F, Al-Inany HG, Repping S et al. (2013) Embryo culture media and IVF/ICSI success rates: a systematic review. Hum Reprod Update 19, Marnet B, Vieitez G, Milhet P, Richoilley G, Lesourd F & Parinaud J. (2000) Computer-assisted assessment of sperm morphology: comparison with conventional techniques. Int J Androl 23, Montjean D, Belloc S, Benkhalifa M, Dalleac A & Menezo Y. (2012) Sperm vacuoles are linked to capacitation and acrosomal status. Hum Reprod 27, Oatway C, Gunby J & Daya S. (2004) Day three versus day two embryo transfer following in vitro fertilization or intracytoplasmic sperm injection. Cochrane Database Syst Rev, CD Oliveira JB, Massaro FC, Baruffi RL, Mauri AL, Petersen CG, Silva LF et al. (2010) Correlation between semen analysis by motile sperm organelle morphology examination and sperm DNA damage. Fertil Steril 94, Parinaud J, Mieusset R, Vieitez G, Labal B & Richoilley G. (1993) Influence of sperm parameters on embryo quality. Fertil Steril 60, Perdrix A, Travers A, Chelli MH, Escalier D, Do Rego JL, Milazzo JP et al. (2011) Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles. Hum Reprod 26, Perdrix A, Saidi R, Menard JF, Gruel E, Milazzo JP, Mace B et al. (2012) Relationship between conventional sperm parameters and motile sperm organelle morphology examination (MSOME). Int J Androl 35, Vanderzwalmen P, Hiemer A, Rubner P, Bach M, Neyer A, Stecher A et al. (2008) Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles. Reprod Biomed Online 17, Wilding M, Coppola G, di Matteo L, Palagiano A, Fusco E & Dale B. (2011) Intracytoplasmic injection of morphologically selected spermatozoa (IMSI) improves outcome after assisted reproduction by deselecting physiologically poor quality spermatozoa. J Assist Reprod Genet 28, World Health Organization (2010) WHO Laboratory Manual for Examination of Human Semen and Semen-Cervical Mucus Interaction, 5th edn. The Press Syndicate of the University of Cambridge, Cambridge, UK American Society of Andrology and European Academy of Andrology Andrology, 2013, 1,