Article High-magnification ICSI overcomes paternal effect resistant to conventional ICSI

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1 RBMOnline - Vol 12. No Reproductive BioMedicine Online; on web 23 November 2005 Article High-magnification ICSI overcomes paternal effect resistant to conventional ICSI André Hazout was co-leader of the ART programme of Frydman s team in Clamart Currently he leads the ART programme of Bichat Hospital in the University Paris VII and the Private Reproductive Medicine and Infertility Unit of Eylau La Muette in Paris. He is Vice President of the French Society of Reproductive Medicine. He was responsible for development of the Hazout oocyte collector and has published extensively in both national and international journals. His recent research interests include the role of somatostatin in the treatment of male infertility, the proteomic profile of the seminal plasma of infertile men, sclerotherapy of endometriosis cysts before ART, and embryo implantation markers in endothelium explants. Dr André Hazout André Hazout 1, Martine Dumont-Hassan 2, Anne-Marie Junca 2, Paul Cohen Bacrie 2, Jan Tesarik 2,3,4 1 ARCEFAR, 15 rue Faraday, Paris; 2 Laboratoire d Eylau, 55 rue Saint-Didier, Paris, France; 3 MARGEN, Molecular Assisted Reproduction and Genetics, Gracia 36, Granada, Spain 4 Correspondence: MARGEN, Molecular Assisted Reproduction and Genetics, Gracia 36, Granada, Spain. Tel: ; Fax: ; cmendoza@ugr.es Abstract Previous studies have shown that repeated intracytoplasmic sperm injection (ICSI) failures can be caused by a paternal effect. Other studies have suggested that ICSI results are compromised if morphologically abnormal spermatozoa are injected into oocytes. This study was undertaken to evaluate the usefulness of a high-magnification optical system to select spermatozoa to be used for ICSI (high-magnification ICSI) in couples with repeated conventional ICSI failures. Couples with two or more previous conventional ICSI failures underwent an additional conventional ICSI attempt, followed by a high-magnification ICSI attempt. The outcomes of the two sequential attempts were compared. In 72 of these patients, sperm DNA integrity was assessed. In the whole group of 125 couples with repeated ICSI failures, high- magnification ICSI improved clinical outcomes (pregnancy, implantation, delivery and birth rates) without affecting biological outcomes (fertilization and cleavage rates, embryo morphology). The improvement of clinical ICSI outcomes was evident both in patients with an elevated degree of sperm DNA fragmentation and in those with normal sperm DNA status. It is concluded that high-magnification ICSI improves clinical outcomes in couples with previous repeated conventional ICSI failures. Keywords: high-magnifi cation sperm selection, ICSI, male factor, paternal effect, sperm DNA damage, TUNEL Introduction Success rates of intracytoplasmic sperm injection (ICSI) were initially thought to be independent of basic sperm parameters (Küpker et al., 1995; Mansour et al., 1995; Nagy et al., 1995; Svalander et al., 1996; Lundin et al., 1997; Sukcharoen et al., 1998). However, later experience has shown that pregnancy and implantation rates are lower in cases in which only morphologically abnormal spermatozoa are available for ICSI, as compared with those in which morphologically normal spermatozoa can be found (De Vos et al., 2003). Moreover, some cases of repeated failure of conventional IVF (Vanderzwalmen et al., 1991; Parinaud et al., 1993; Janny and Menezo, 1994) and ICSI (Hammadeh et al., 1996; Sanchez et al., 1996; Shoukir et al., 1998) were suggested to be caused by a paternal effect on the early embryo development, and this hypothesis has recently been confirmed in a shared oocyte donation model (Tesarik et al., 2002, 2004; Tesarik, 2005). In fact, embryos developing from donor oocytes fertilized by spermatozoa of certain patients repeatedly failed to yield a clinical pregnancy, whereas high implantation, pregnancy and birth rates were achieved with sibling oocytes from the same donors that were injected with spermatozoa from patients undergoing their first ICSI attempt (Tesarik et al., 2002, 2004). The accuracy with which the morphological normality of spermatozoa used for ICSI can be assessed is dependent on the resolving power of the optical magnification system used. Conventionally, ICSI is performed with the use of a 20 objective, resulting in an overall optical magnification of about 400 (De Vos et al., 2003). However, spermatozoa appearing as morphologically normal at this magnification may in fact carry various structural abnormalities that can only be detected with the use of higher optical magnifications than those usually 19

2 20 employed for sperm selection for ICSI (Bartoov et al., 2002). A prospective controlled study, performed in couples with male infertility and at least two previous failed ICSI attempts, showed that ICSI with spermatozoa selected at a digitally enhanced magnification of 6000 resulted in a significantly higher pregnancy rate as compared with conventional ICSI (Bartoov et al., 2003). This was achieved by using a 100 oilimmersion objective and a secondary magnification system (Bartoov et al., 2003). This system makes it possible to detect, in the living state, most of the sperm anomalies recognized by the conventional basic sperm analysis (World Health Organization, 1999) performed on fixed and stained sperm samples. A subsequent study, performed by the same group, showed that higher implantation and pregnancy rates were achieved by ICSI when at least some morphologically normal spermatozoa could be selected with this high-power system (Berkovitz et al., 2005). However, high numbers (three or four) of embryos were transferred in both studies, leading to multiple pregnancy rates of 48.4 and 40% respectively (Bartoov et al., 2003; Berkovitz et al., 2005). This study reports a series of cases, characterized by at least two previous conventional ICSI failures, in which highmagnification ICSI was applied in the context of an embryo transfer policy limiting the number of embryos per transfer to two unless only embryos with poor morphological quality were available. Special attention is paid to a subgroup of this series in which sperm DNA integrity was assessed and the outcomes of high-magnification ICSI could be compared in cases with different degrees of sperm DNA fragmentation. Materials and methods Patients and design This study involved 125 couples who met the following criteria: two or more previous ICSI attempts that failed to establish an ongoing pregnancy, normal ovarian reserve, the absence of endometriosis, and the female age of <38 years. Eighty-eight of the couples presented with male factor, according to the World Health Organization (WHO) criteria (World Health Organization, 1999). Twenty-four of these men had moderate teratozoospermia (11 27% of normal forms), 49 had severe teratozoospermia (1 10% of normal forms) associated with oligoasthenozoospermia and 15 showed oligoasthenozoospermia with normal sperm morphology. Male partners of the remaining 37 couples had normal semen. These evaluations were made on Papanicolaoustained smears prepared according to the WHO criteria (World Health Organization, 1999), but applying particular limits for moderate and severe teratozoospermia (see above). Seventy-two of the men involved in this study were examined for sperm DNA integrity. This was not applied to all couples, as some were treated before evaluation of DNA integrity became standard policy for all couples with repeated ICSI failures. Of the 72 examined, 21 showed an elevated incidence of sperm nuclear DNA fragmentation (see below). Within the subgroup of 72 cases with sperm DNA integrity assessment, increased incidence of sperm DNA fragmentation was associated with moderate teratozoospermia in five cases (6.9%), with severe teratozoospermia in 10 cases (13.9%), with oligoasthenozoospermia and normal sperm morphology in three cases (4.2%) and with normal basic semen parameters in three cases (4.2%). Sperm evaluation Basic sperm characteristics were assessed according to the WHO guidelines (World Health Organization, 1999). Sperm DNA fragmentation was evaluated by terminal deoxyribonucleotidyl transferase-mediated dutp fluoresceindutp nick-end labelling (TUNEL) followed by counting labelled and unlabelled spermatozoa by flow cytometry using an Epics XL-MCL (Beckman Coulter Inc., Fullerton, CA, USA) cell sorter. The TUNEL reaction was performed with the In-Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany), and spermatozoa were processed as recommended by the manufacturer. Briefly, ejaculated spermatozoa were washed from seminal plasma in phosphatebuffered saline (PBS) min after ejaculation, fixed in 2% paraformaldehyde in PBS at laboratory temperature for 1 h and permeabilized with 0.1% Triton X in 1% sodium citrate. Fixed and permeabilized sperm suspensions were incubated with the TUNEL reaction mix at 37ºC for 1 h, washed in PBS and analysed in the cell sorter with the use of the Sperm TUNEL programme. For each test, a negative control (omitting the enzyme terminal deoxyribonucleotidyl transferase from the reaction mixture) and a positive control (spermatozoa preincubated with 1 mg/ml DNase at laboratory temperature for 20 min) were performed. Only samples with a sperm count of 600,000 were processed. The incidence of sperm DNA fragmentation was considered normal when the percentage of DNA-fragmented spermatozoa was <30%, moderate when the percentage of DNA-fragmented spermatozoa was 30 40% and high when the percentage of DNA-fragmented spermatozoa was >40%. These cut-off values were based on a preliminary series of observations comparing ICSI outcomes in patients with different degrees of sperm DNA fragmentation assessed with the use of the above methods. Ovarian stimulation and oocyte recovery Ovarian stimulation was performed with the use of recombinant human FSH (Gonal-f; Serono, Geneva, Switzerland) after pituitary down-regulation with triptorelin (Decapeptyl; Ipsen Pharma, Paris, France) started in the mid-luteal phase. The guidelines of this protocol have been published previously (Hazout et al., 2004). When at least two follicles had reached a mean diameter of 18 mm, ovulation was induced by subcutaneous administration of 250 μg recombinant human human chorionic gonadotrophin (HCG: Ovitrelle; Serono). Oocytes were recovered by ultrasound-guided transvaginal follicle aspiration 36 h after HCG injection. Conventional ICSI Conventional ICSI was performed with the use of an Olympus IX 70 inverted microscope equipped with Narishige 231 D-2 (Narishige, Tokyo, Japan) remote control hydraulic micromanipulators and Narishige IM-9B injectors. Both sperm

3 selection for ICSI and the ICSI procedure itself were carried out with a 20 objective lens (Figure 1A). High-magnification ICSI Spermatozoa for high-magnification ICSI (Figure 1B) were selected with the use of a 100 oil-immersion objective lens and an Olympus IX 70 inverted microscope equipped with Nomarski differential interference contrast optics, as described previously (Bartoov et al., 2003; Berkovitz et al., 2005). Sperm selection was made at laboratory temperature in drops of salt solution containing hyaluronate and human serum albumin (Sperm Catch; Nidacon, Lyon, France). The drops were covered with sterile mineral oil (Cryo Biosystems, L Aigle, France). The number of spermatozoa selected for each highmagnification ICSI attempt was twice that of mature oocytes available for the given attempt. Only motile spermatozoa classified as morphologically normal and lacking intranuclear vacuoles were selected. This selection step took 30 min to 2 h, depending on the degree of sperm morphology impairment in each case. After the sperm selection step, oocytes together with the selected spermatozoa were placed in an Olympus IX 70 inverted microscope equipped with Hofman modulation contrast optics, and ICSI was performed with the use of a 20 objective lens as for conventional ICSI. Oocyte and embryo culture, embryo grading and transfer Sperm-injected oocytes, zygotes and embryos were cultured at 37ºC IVF-30 medium (Vitrolife, Gothenberg, Sweden) equilibrated with 5% CO 2 in air. Fertilization was assessed h after conventional or high-magnification ICSI. Zygotes showing two equal-sized pronuclei were cultured further, under the same conditions, for an additional 2 days. Embryos were graded on days 2 and 3 after ICSI according to previously published criteria (Tesarik and Greco, 1999; Tesarik et al., 2000). On day 3 after ICSI, two best-scoring embryos were transferred to the patient s uterus. Exceptionally, when only low-grade embryos were available, three embryos were transferred. Statistical analysis Differences between groups were assessed by two-tailed chi-squared test with Yates correction or Fisher s exact test for categorical variables, and by Mann Whitney U-test - for continuous variables. All analyses were performed using the Statistica 5.0 package (Statsoft Version 5.1, Hamburg, Germany). Results All patients having at least two previous unsuccessful ICSI attempts, excluding those with diminished ovarian reserve, endometriosis, and female age of 38 years (see Materials and methods), were included. All the patients included continued throughout the study and were analysed. The two sequential ICSI attempts, one performed by conventional ICSI and the other by high-magnification ICSI in 125 couples, did not differ as to the basic ovarian stimulation cycle characteristics, including the duration of stimulation, the peak serum oestradiol concentration, and the numbers of total and metaphase II (MII) oocytes recovered (Table 1). With similar numbers of oocytes injected in the two sequential attempts, both conventional and high-magnification ICSI resulted in comparable yields of two-pronucleated zygotes, cleaved embryos and good-morphology embryos (Table 2). However, the clinical outcomes differed significantly between the two attempts. High-magnification ICSI resulted in higher clinical pregnancy (37.6 versus 2.4%, P < 0.001), clinical implantation (20.3 versus 0.8%, P < 0.001) delivery (33.6 versus 0%, P < 0.001) and birth (17.6 versus 0%, P < 0.001) rates as compared with the last conventional ICSI attempt in the same couples (Table 3). In a subgroup (n = 72) of patients involved in this study, the percentage of ejaculated spermatozoa with nuclear DNA fragmentation was evaluated by TUNEL. When the clinical outcomes of conventional and high-magnification ICSI were compared separately in this subgroup of patients, a marked increase in clinical implantation and birth rates was observed in patients with both normal (<30%), moderately (30 40%) and highly (>40%) increased percentage of DNA-fragmented spermatozoa in the ejaculate (Table 4). Figure 1. Comparison of the resolving power of the sperm selection systems used in conventional intracytoplasmic sperm injection (ICSI) (A) and high-magnification ICSI (B). Magnification: A, x400; B, x

4 Table 1. Comparison of basic ovarian stimulation cycle characteristics in the last conventional intracytoplasmic sperm injection (ICSI) cycle and the subsequent ICSI attempt with high-magnification sperm selection. ICSI attempt Characteristic Conventional High magnification Duration of stimulation (days) 11.8 ± ± 0.4 Peak serum oestradiol concentration (pg/ml) 2197 ± ± 498 No. of oocytes recovered a 11.2 ± ± 2.7 No. of MII oocytes recovered a 9.5 ± ± 2.5 Values are means ± SD. a Per stimulated cycle. Table 2. Comparison of fertilization outcome variables between the last conventional intracytoplasmic sperm injection (ICSI) attempt and the subsequent ICSI attempts with high-magnification sperm selection. ICSI attempt Outcome variable Conventional High magnifi cation Oocytes injected 9.5 ± ± 2.5 2PN zygotes 6.2 ± ± 1.8 Cleaved embryos 5.9 ± ± 1.0 Good-morphology embryos 3.1 ± ± 1.2 Values are means ± SD per cycle. PN = pronucleate. Table 3. Comparison of clinical outcome variables between the last conventional intracytoplasmic sperm injection (ICSI) attempt and the subsequent ICSI attempts with high-magnification sperm selection. Outcome variable ICSI attempt P-value Conventional Highmagnifi cation Transfer procedures (n) Embryos transferred (n) >0.05 Total pregnancies (n) 8 51 <0.001 Clinical pregnancies (n) 3 47 <0.001 Deliveries (n) 0 42 <0.001 Gestational sacs with heartbeat (n) 2 53 <0.001 Babies born (n) 0 46 <0.001 Pregnancy rate (%) <0.001 Clinical pregnancy rate (%) <0.001 Clinical implantation rate (%) <0.001 Delivery rate (%) <0.001 Birth rate (%) <

5 Table 4. Comparison of clinical outcome variables between the last conventional intracytoplasmic sperm injection (ICSI) attempt and the subsequent ICSI attempts with high-magnification sperm selection in patients (n = 72) with different degrees of sperm DNA fragmentation. % DNA-fragmented spermatozoa <30% (n = 51) 30 40% (n = 11) >40% (n = 10) ICSI attempt Implantation Birth Implantation Birth Implantation Birth rate a rate b rate a rate b rate a rate b Conventional (%) 1/107 (0.9) 0/107 (0) 0/25 (0) 0/25 (0) 1/24 (0.7) 0/24 (0) High-magnification (%) 25/106 (23.6) 20/106 (18.9) 4/23 (17.4) 4/23 (17.4) 7/21 (33.3) 6/21 (28.6) P-value <0.001 <0.001 <0.05 <0.05 <0.01 <0.01 a Number of gestational sacs per number of embryos transferred. b Number of babies born per number of embryos transferred. Discussion The results of this clinical observational study show that the recourse to high-magnification selection of spermatozoa to be used for ICSI leads to significantly higher (P < 0.001) clinical pregnancy and implantation rates as compared with the last conventional ICSI attempt performed in the same group of couples selected on the basis of a history of at least two previous consequent conventional ICSI cycles without pregnancy. In spite of the fact that this study was not designed as randomized and placebo-controlled, the difference between the clinical outcomes of the last conventional ICSI attempt and the subsequent high-magnification ICSI attempt was so marked that it could not be attributed to biological variability in the quality of gametes. This conclusion is also substantiated by the similarity of the basal ovarian stimulation cycle characteristics and of the yield of M II oocytes recovered in the two sequential attempts. In a subgroup of patients involved in this study, the degree of sperm DNA fragmentation was determined. However, this test was not performed directly with the sperm samples used for ICSI in the two sequential attempts. Even though a previous prospective randomized study (Greco et al., 2005a) showed that a spontaneous, treatment-independent improvement of sperm DNA fragmentation figures can occur with time, this was counterbalanced by a spontaneous impairment in other patients (Greco et al., 2005a). The number of cases involved in the present study was high enough to absorb the effects of such fortuitous variations on the whole group outcomes. DNA damage cannot be directly evaluated in spermatozoa selected for high-magnification ICSI. However, the resolving power offered by this system makes it possible to exclude spermatozoa with small-sized intranuclear vacuoles which would not be detected in the conventional ICSI setting. The presence of such vacuoles may reflect molecular defects responsible for abnormal chromatin remodelling during sperm maturation (Cayli et al., 2003) which, in its turn, may render spermatozoa more vulnerable to DNA damage caused by externally or internally produced reactive oxygen species (Sakkas et al., 2003; Agarwal et al., 2005). The exclusion of such spermatozoa can thus be expected to decrease the probability of accidental injection of a DNA-damaged spermatozoon to the oocyte. Interestingly, the improvement of clinical outcomes by highmagnification ICSI observed in this study was not accompanied by an improvement in biological outcome characteristics reflecting sperm fertilizing ability and the quality of the early post-fertilization development. In fact, fertilization rate, embryo cleavage rate and embryo morphology grade were similar in the conventional ICSI attempts and in the high-magnification ICSI attempts. In agreement with this observation, an improvement in clinical outcomes without appreciable changes in fertilization and embryo cleavage parameters has also been noted in two recent studies in which the negative impact of ejaculated sperm DNA fragmentation on embryo implantation and pregnancy was alleviated by recourse to testicular spermatozoa (Greco et al., 2005b) or by performing ICSI with ejaculated spermatozoa after 2 months of oral antioxidant treatment (Greco et al., 2005c). As shown previously, sperm nuclear DNA fragmentation is closely related to male-derived repeated ICSI failures, and it is also the only marker of such failures that can be objectively and reproducibly diagnosed nowadays (Tesarik et al., 2004; Tesarik, 2005). However, many other cases of male-derived repeated ICSI failure are not associated with sperm DNA damage. By using a sibling oocyte model based on a donor oocyte sharing programme, two types of adverse paternal effects on early embryo development have been distinguished an early paternal effect, which impairs post-fertilization development from as early as the pronuclear zygote stage, and a late paternal effect, which disturbs implantation without producing visible alterations of embryo cleavage speed and morphology grade (Tesarik et al., 2002, 2004; Tesarik, 2005). However, a relationship between sperm DNA fragmentation and embryo quality has also been reported (Henkel et al., 2003). It was also noted that the late paternal effect, but not the early one, is associated with increased sperm DNA fragmentation (Tesarik et al., 2004). In the present study, sperm DNA integrity was assessed in a subgroup of the cases involved. The data obtained show an increased percentage of DNAfragmented spermatozoa in only 29.2% of these cases. By extrapolation, it can thus be expected that sperm DNA fragmentation was not the main cause of repeated ICSI failure in most of the cases involved in this study. According to the previously suggested classification (Tesarik et al., 2004; Tesarik, 2005), most of the present cases would thus fall into 23

6 24 the category of early paternaleffect. Therefore, it is possible that high-magnification ICSI is useful in both the early and the late paternal effect conditions. The fact that no differences in embryo grade between conventional and high-magnification ICSI could be demonstrated in this study suggests that the use of high-magnification sperm selection makes it possible to improve embryo implantation potential without affecting conventional parameters of preimplantation embryo development as assessed by non-invasive microscopical evaluation. Alternatively, the small trend towards a better embryo morphology grade in the high-magnification ICSI group observed in this study may reach statistical significance in higher-powered studies. Recent studies have suggested that neither sperm morphology (McKenzie et al., 2004) nor sperm DNA fragmentation (Huang et al., 2005) by itself may be critical for ICSI clinical outcome if the overall clinical context is not taken into account. Thus, it is important to emphasize that the first selection criterion for the inclusion of patients in the present study was the repeated ICSI failure in previous cycles. It remains to be determined how results of sperm morphology and DNA fragmentation should be best interpreted with regard to the number of previous failed ICSI attempts. These data suggest that high-magnification ICSI is especially useful in cases of paternally caused repeated ICSI failure in which a pathologically increased percentage of DNAfragmented spermatozoa is found (late paternal effect), but also in those with normal values of sperm DNA fragmentation (early paternal effect). A prospective study is needed to compare the benefits of this technique in these two conditions. References Agarwal A, Gupta S, Sharma RK 2005 Oxidative stress and its implication in female infertility a clinician s perspective. 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7 Percoll gradient centrifugation and swim-up procedures. Human Reproduction 6, World Health Organization 1999 WHO Laboratory Manual for the Examination of Human Semen and Sperm Cervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge. Received 25 August 2005; refereed 24 September 2005; accepted 25 October