The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle

Reproductive BioMedicine Online (2012) 24, 521 526 www.sciencedirect.com www.rbmonline.com ARTICLE The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle Markus Montag a,b, *, Maria Köster b, Katrin van der Ven b, Ulrike Bohlen b, Hans van der Ven b a Department of Gynecological Endocrinology and Fertility Disorders, University of Heidelberg, Voßstr. 9, 69115 Heidelberg, Germany; b Department of Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Bonn, Germany * Corresponding author. E-mail address: markus.montag@med.uni-heidelberg.de (M Montag). Prof Markus Montag obtained his PhD at the German Cancer Research Centre and worked at NUH, Singapore with Prof SC Ng. Returning from Singapore, he was laboratory director in a private IVF unit before becoming Director of the Reproductive Biology Laboratory at the University Clinics of Bonn in 1995 and Associate Professor for Experimental Reproductive Medicine in 2009. In 2011 he became Head of the IVF Laboratory at the University Clinics of Heidelberg. He is actively involved in counselling IVF centres and educating young people in the field worldwide. His research areas include the use of lasers, ovarian tissue cryobanking, oocyte activation, polarization microscopy and polar body biopsy. Abstract Following intracytoplasmic sperm injection (ICSI), some patients present low or zero fertilization rates. Artificial oocyte activation has been proposed as a suitable means to overcome this problem. This study applied artificial oocyte activation in patient cohorts with a history of no fertilization (0%, group 1), fertilization between 1 and 29% (group 2) or fertilization between 30 and 50% (group 3) in initial ICSI cycles. In the following treatment cycles, oocytes were activated after ICSI using calcium ionophore. Fertilization, pregnancy and take-home baby rates were compared with the previous cycle without activation. In group 1, fertilization rate was 41.6%, embryos for transfer were available in 82.1% of cycles, giving a clinical pregnancy rate of 18.8% and take-home baby rate of 12.8%. In group 2, despite a lower transfer rate (87.9% versus 100%, P < 0.05), there were higher fertilization and clinical pregnancy rates (44.4% versus 19.3% and 31.4% versus 12.8%, respectively, P < 0.05) and take-home baby rate was 24.1% versus 12.8%. In group 3, fertilization rates differed (56.1% versus 36.8%; P < 0.001) but all other parameters were similar. Artificial oocyte activation has great potential especially in patients showing compromised fertilization rates below 30% after standard ICSI. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: activation failure, fertilization, ionophore, oocyte, spermatozoa 1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2012.02.002

522 M Montag et al. Introduction The introduction of intracytoplasmic sperm injection (ICSI) has made it possible to overcome most problems associated with male factor infertility, namely low sperm count and reduced motility (Palermo et al., 1992, 1996). Since then, ICSI has proven to be very efficient following fertilization failure in a normal IVF cycle and to yield constantly high fertilization rates of 70 80% on average. However, although failed fertilization after ICSI is a rare event (Liu et al., 1995), it still exists. Moreover, 0 30% fertilization rates can consistently be observed in repeated ICSI cycles for some patients. The incidence of fertilization problems after ICSI raises the question of the underlying aetiology, especially with regard to the role of the spermatozoon for achieving successful fertilization. One of the initial events of the fertilization cascade is the activation of the oocyte by a sperm-derived oocyte activation factor. The exact nature of this sperm factor was among the focus of basic research since the early 1990s (Kline and Kline, 1992; Swann and Ozil, 1994). It finally resulted in the identification of a novel sperm-specific phospholipase PLC, PLCf, which is considered to be responsible for mammalian oocyte activation (Saunders et al., 2002; Swann et al., 2004). To date, two studies have shown that fertilization failure in infertile men can be linked to aberrant forms or reduced amounts or even absence of PLCf (Heytens et al., 2009; Yoon et al., 2008). The majority of patients in these studies were diagnosed with round-headed spermatozoa or globozoospermia, a sperm defect which is associated with failed oocyte activation after ICSI (Battaglia et al., 1997; Rybouchkin et al., 1996). One successful ICSI treatment option for men with globozoospermia is artificial oocyte activation using calcium ionophore (Rybouchkin et al., 1997; Taylor et al., 2010). The potential of calcium ionophore to support oocyte activation and yield high fertilization rates was shown at the beginning of the era of ICSI (Tesarik et al., 1995). However, artificial activation was never routinely applied after ICSI but instead was restricted to those patients showing fertilization failure after ICSI, independently of the sperm diagnosis (Eldar-Geva et al., 2003; Heindryckx et al., 2008, 2005). In 2003, this study centre started applying artificial oocyte activation in cases with 0%, 1 29% and 30 50% fertilization after ICSI. Here is presented a retrospective analysis of the success rates in these cases by comparing the outcomes of the initial cycles with the following cycles that applied artificial oocyte activation. Materials and methods Patients, ovarian stimulation and oocyte retrieval In the study period between 2003 and 2009, patients with 0%, 1 29% or 30 50% fertilization in a previous ICSI cycle were considered for this study. Ethical approval was not required as the study was considered as a therapeutic treatment approach based on published reports (Eldar-Geva et al., 2003; Rybouchkin et al., 1997; Tesarik et al., 1995). Cases with surgically retrieved spermatozoa from the testis or the epididymis were excluded. Patients gave informed consent for artificial oocyte activation. Ovarian stimulation and oocyte retrieval followed standard protocols using either long or short stimulation with agonist or antagonist. Ovulation was triggered by administration of 250 lg human chorionic gonadotrophin (HCG) and oocyte retrieval was scheduled 32 34 h later. ICSI, culture, embryo transfer and pregnancy Cumulus oocyte complexes were isolated and rinsed in HEPES-buffered medium (gamete medium; Cook, Brisbane, Australia) followed by fertilization medium (Cook) and incubated in fertilization medium in humidified air at 6% CO 2 in a 4-well dish. After 2 3 h, oocytes were denuded with hyaluronidase (Cook) and incubated for another 1 2 h prior to ICSI in cleavage medium (Cook). ICSI was performed at an inverted microscope (DMIRB; Leica, Wetzlar, Germany) equipped with a heated table and micromanipulators (Narishige, Tokyo, Japan). Spermatozoa were prepared by a modified mini-swim-up technique (Montag et al., 1997) and placed into polyvinylpyrrolidone solution (ICSI medium; Cook) in a standard plastic dish. In the same dish, oocytes were placed in droplets of gamete medium and were injected using appropriate micro-capillaries (Cook) with a standard ICSI injection technique, as described (Montag et al., 1997). The presence of pronuclei was checked 16 20 h after ICSI. Two to three 2-pronuclear (PN) oocytes were chosen for further culture and transfer, and surplus 2PN oocytes were cryopreserved. 2PN oocytes for transfer and culture were placed into a new dish and were cultured for another 1 2 days. Embryo transfer was performed on day 3 as described (Montag et al., 2002). A first bhcg test was performed 14 days after oocyte retrieval and if positive the test was repeated 2 days later. The presence and number of fetal sacs was confirmed by ultrasound scan 14 days later and fetal heart beat was investigated another 14 days later. The implantation rate was calculated by the number of fetal sacs divided by the number of embryos transferred. Only pregnancies with a proven implantation were classified as clinical pregnancies. Pregnant patients were asked to submit data on birthweight and the health of the children born. Artificial oocyte activation Oocyte activation was performed as described (Tesarik et al., 1995) with some modifications. Immediately after ICSI, oocytes were incubated for 15 min in a 50-ll droplet of activation solution (10 lmol/l calcium ionophore A23187 in culture medium; cleavage medium; Cook) covered by mineral oil (Cook). After exposure to the activation solution, oocytes were thoroughly washed in culture medium and incubated under standard conditions in 6% CO 2,5%O 2 and 89% N 2 in humidified atmosphere (Minc; Cook). Statistics Statistical analysis used GrapPadPrism 5 and ANOVA or chi-squares tests whenever appropriate. P-values <0.05 were considered significant.

Artificial oocyte activation 523 Results Artificial activation in patients with 0% initial fertilization rate A total of 27 patients, who presented with absent fertilization in 31 standard ICSI cycles, returned in the study period for at least one more ICSI cycle in which artificial oocyte activation was performed. When the characteristic features of the 31 cycles without activation to the 39 cycles with artificial activation were compared, there was a non-significant increase in the mean female age (36.1 years versus 37.8 years; Table 1). The number of oocytes retrieved, as well as the number of mature oocytes for ICSI, was different (5.4 and 3.7 versus 7.4 and 5.9, P < 0.05 and P < 0.001, respectively). However, following artificial oocyte activation, a fertilization rate of 41.6% was achieved, which resulted in 82.1% embryo transfers with a mean number of 1.46 embryos transferred. The implantation rate was 12.3%, giving rise to a positive bhcg rate, clinical pregnancy rate and take-home baby rate per transfer cycle of 25.0%, 18.8% and 15.6%, respectively. The birthweight of the children born was 3196 ± 340 g and all children born were healthy and without any malformations. Artificial activation in patients with 1 29% initial fertilization rate A total of 38 patients presented with a fertilization rate of 1 29% in 47 standard ICSI cycles and returned in the study period for at least one more ICSI cycle, in which artificial oocyte activation was performed. When the characteristic features of the 47 cycles without activation were compared with the 58 cycles with artificial activation, there was a significant increase in the mean female age (33.3 years to 35.3 years, P < 0.05; Table 2). The number of oocytes retrieved, as well as the number of mature oocytes for ICSI, was not different (8.0 and 6.2 versus 7.8 and 6.1, respectively). In the cycles with artificial oocyte activation, the fertilization rates were higher (19.3% versus 44.4%, P < 0.001). However, there was a lower rate of embryo transfer cycles (100% versus 87.9%, P < 0.05), because in six patients who had only one oocyte fertilized in the previous cycles without activation, no fertilization occurred in seven treatment cycles with activation. A comparable mean number of embryos were transferred after activation (1.48 versus 1.57). The positive bhcg rate as well as the clinical pregnancy rate was significantly higher with artificial oocyte activation than without (14.9% and 12.8% versus 37.3% and 31.4%, respectively, P < 0.05). There was a non-significant trend towards higher implantation rates in artificial activation cycles (8.6% versus 17.6%). There was also no significant difference in the abortion rate (0.0% versus 12.5%) and the take-home baby rate per embryo transfer (12.8% versus 27.5%). With artificial oocyte activation the take-home baby rate per ICSI cycle was higher (12.8% versus 24.1%) although the difference did not reach statistical significance. The birthweight of the children born was not different (3445 ± 399 g with activation versus 3952 ± 530 g without artificial activation). All children born were healthy and without any malformations. Artificial activation in patients with 30 50% initial fertilization rate In the same time period, a total of 24 patients presented with 30 50% fertilization rate in 25 standard ICSI cycles and consented to a subsequent treatment with artificial oocyte activation in another 32 ICSI cycles. The mean female age was 34.0 years in the standard cycles and 35.4 years in the cycles with activation (Table 3). The mean number of oocytes retrieved and injected was not different between standard and artificial activation cycles (9.2 and 7.0 versus 8.9 and 6.7, respectively). Fertilization rates Table 1 Results of artificial oocyte activation in patients with 0% fertilization rates in an initial cycle (group 1). Standard ICSI Artificial oocyte activation P-value Patients (n) 27 27 Female age (years) 36.1 ± 4.2 37.8 ± 3.4 NS ICSI cycles (n) 31 39 Oocytes retrieved 5.4 ± 2.8 7.4 ± 3.8 <0.05 Oocytes injected 3.7 ± 2.4 5.9 ± 3.5 <0.001 Fertilization/injected oocyte 0 (0/114) 41.6 (96/231) a <0.05 ET cycles 0 (0/31) 82.1 (32/39) <0.05 Embryos transferred (mean) 0 1.46 <0.05 Positive bhcg/et 0 25.0 (8/32) <0.05 Clinical pregnancy/et 0 18.8 (6/32) <0.05 Implantation/embryos transferred 0 12.3 (7/57) <0.05 Abortion/clinical pregnancy 0 16.7 (1/6) <0.05 Take-home baby/et 0 15.6 (5/32) <0.05 Take-home baby/cycle 0 12.8 (5/39) <0.05 Values are mean ± SD or % (n/total), unless otherwise indicated. ET = embryo transfer; NS = not significant. a No fertilization occurred in seven patients in seven activation cycles.

524 M Montag et al. Table 2 Results of artificial oocyte activation in patients with 1 29% fertilization rates in an initial cycle (group 2). Standard ICSI Artificial oocyte activation P-value Patients (n) 38 38 Female age (years) 33.3 ± 4.4 35.3 ± 3.7 <0.05 ICSI cycles (n) 47 58 Oocytes retrieved 8.0 ± 4.0 7.8 ± 4.1 NS Oocytes injected 6.2 ± 3.6 6.1 ± 3.5 NS Fertilization/injected oocyte 19.3 (72/373) 44.4 (161/363) a <0.001 ET cycles 100.0 (47/47) 87.9 (51/58) <0.05 Embryos transferred (mean) 1.48 1.57 NS Positive bhcg/et 14.9 (7/47) 37.3 (19/51) <0.05 Clinical pregnancy/et 12.8 (6/47) 31.4 (16/51) <0.05 Implantation/embryos transferred 8.6 (6/70) 17.6 (16/91) NS Abortion/clinical pregnancy 0 (0/6) 12.5 (2/16) NS Take-home baby/et 12.8 (6/47) 27.5 (14/51) NS Take-home baby/cycle 12.8 (6/47) 24.1 (14/58) NS Values are mean ± SD or % (n/total), unless otherwise indicated. ET = embryo transfer; NS = not significant. a No fertilization occurred in six patients in seven activation cycles. These patients had only one oocyte fertilized in the previous cycles without activation. Table 3 Results of artificial oocyte activation in patients with 30 50% fertilization rates in an initial cycle (group 3). Standard ICSI Artificial oocyte activation P-value Patients (n) 24 24 Female age (years) 34.0 ± 4.9 35.4 ± 4.3 NS ICSI cycles (n) 25 32 Oocytes retrieved 9.2 ± 3.7 8.9 ± 4.7 NS Oocytes injected 7.0 ± 2.9 6.7 ± 3.3 NS Fertilization/injected oocyte 36.8 (64/174) 56.1 (124/221) <0.001 ET cycles 100 (25/25) 90.6 (29/32) NS Embryos transferred (mean) 1.84 1.96 NS Positive bhcg/et 28.0 (7/25) 37.9 (11/29) NS Clinical pregnancy/et 16.0 (4/25) 27.6 (8/29) NS Implantation/embryos transferred 8.7 (4/46) 17.5 (10/57) NS Abortion/clinical pregnancy 50.0 (2/4) 25.0 (2/8) NS Take-home baby/et 8.0 (2/25) 20.7 (6/29) NS Take-home baby/cycle 8.0 (2/25) 18.8 (6/32) NS Values are mean ± SD or % (n/total), unless otherwise indicated. ET = embryo transfer; NS = not significant. were significantly higher following artificial oocyte activation (36.8% versus 56.1%, P < 0.001); however, there was no difference in cycles with embryo transfer (100% versus 90.6%) as well as the mean number of embryos transferred (1.84 versus 1.96). Following artificial activation, positive bhcg rates (28.0% versus 37.9%), clinical pregnancy rates per transfer (16.0% versus 27.6%), implantation rates (8.7% versus 17.5%), abortion rates (50.0% versus 25.0%) and take-home baby rate per transfer (8.0% versus 20.7%) or per ICSI cycle (8.0% versus 18.8%) were in favour of the activation group but did not reach significance due to low numbers. All children born were healthy and no malformations were reported. Discussion Artificial oocyte activation was proposed at the beginning of the ICSI era for improving the overall success rates (Sousa

Artificial oocyte activation 525 and Tesarik, 1994). However, with time ICSI proved to be a robust technique with reproducible and comparable results among different clinics (Palermo et al., 1996), especially in view of the fertilization rates. Failure of fertilization after ICSI was initially reported for globozoospermic patients presenting with round-headed spermatozoa (Rybouchkin et al., 1996). The first event in the fertilization cascade is the activation of the oocyte through a sperm-derived oocyte activation factor (Kashir et al., 2010). This factor is considered to be an isoform of phospholipase C, PLCf (Saunders et al., 2002). Several publications report on the absence of PLCf in males with failed fertilization and the majority of these presented with globozoospermia (Heytens et al., 2009; Yoon et al., 2008). Although patients can be tested for the presence or absence of PLCf, other factors may also be involved. This may also comprise activation-related deficiencies in the oocyte or the spermatozoon and changes in the activation cascade which are not yet known as being clinically relevant. The potential of a spermatozoon from a patient to activate oocytes can be investigated by the injection of a spermatozoon or a sperm extract into mouse oocytes and the related mouse oocyte activation test (MOAT) has been used (Rybouchkin et al., 1996). When this test was applied in patients with severe oligoteratozoospermia or with testicular spermatozoa, a high rate of failed oocyte activation was noted (Heytens et al., 2009; Kyono et al., 2008; Tesarik et al., 2002). Although MOAT is a reliable indicator for oocyte activation problems in a subsequent ICSI treatment cycle (Heindryckx et al., 2005), it cannot be applied routinely in every case of fertilization failure or every assisted reproduction unit. Nevertheless, fertilization rates below 50% in repeated ICSI cycles in the same patient can be due to impaired oocyte activation probably caused by a sperm deficiency. This study was conducted on patients who had a first failed ICSI cycle with fertilization rates below 50% and returned for another intervention cycle. The initial failed cycle served as a control. This has implications towards the validity of using such control data for pregnancy rates, as patients achieving pregnancy in a first cycle will probably not return. However, it will not affect activation-induced changes in fertilization rates and hence the patients were grouped according to the fertilization rates in the initial cycles. These data show that oocyte activation has a significant benefit when applied to patients with completely failed fertilization or when fertilization rates were below 30% in the initial ICSI cycle. For the patient cohort with complete fertilization failure in a first cycle, there was an overall change in the stimulation protocol. However, there was not a standard shift from one protocol to another, which would allow for a general statement regarding a possible effect on oocyte activation capacity. These changes probably had an impact on the improvement of the cycle outcome, as more oocytes were available for the subsequent ICSI cycles. However, stimulation protocols were not different in the patient cohort with a fertilization rate of 1 29% in the initial cycle. Subsequent treatment cycles with activation showed no differences for oocyte numbers, and transfer of a comparable mean number of embryos resulted in significantly higher fertilization, biochemical and clinical pregnancy rates. In patients with fertilization rates in the range of 30 50%, there was a significant increase in the fertilization rate but all other investigated parameters did not reach significance. This could be due to the small sample size. However, despite a trend in favour of pregnancy, implantation and take-home baby rates, the success rates for the control group in the 30 50% cohort show that these patients probably did not require activation at all. In view of the activation being another intervention, it is questionable if it should be recommended as a standard treatment in patients with 30 50% fertilization rates. Artificial activation by calcium ionophore causes only a single calcium wave in oocytes and does not lead to calcium oscillations. Therefore, the oocyte still plays a major role in converting this initial calcium peak into an activation-sufficient event. Mimicking calcium oscillations would probably be more critical, as one study showed that a non-physiological oscillation pattern may disturb early embryo development (Ozil et al., 2005). Artificial oocyte activation can overcome the problem of failed (0%) or very low (1 29%) fertilization and it has been also applied by others to patients with normo- or oligozoospermia who otherwise have no underlying disease or other symptoms. These studies showed an improvement in fertilization rates which in some patients even reached normal levels (Heindryckx et al., 2008, 2005; Kyono et al., 2008; Yanagida et al., 2006). As far as is known, the current study is the first to investigate the effect of artificial oocyte activation in relation to the degree of fertilization in the previous ICSI cycle. However, despite the significant increase in fertilization rates, the values reported here do not reach the normal range of more than 70% as reported by others (Heindryckx et al., 2008, 2005) but this may be due to differences in the patient cohort or by the use of an activation protocol which initiates only a single calcium rise instead of calcium oscillations. In the patient cohort of this study, there were no cases of globozoospermia. Therefore reduced fertilization although considered a rare event after ICSI may reveal an aetiology of infertility which still awaits a detailed characterization. This study noted in some treatment cycles in groups 1 and 2 that the application of artificial activation did not improve the results and some patients even received no transfer after activation. However, in these cycles the number of oocytes available for injection was close to 2 3 and thus it is difficult to say if these patients had a real sperm activation deficiency or if the activation procedure failed due to an oocyte problem or the low number of oocytes available. Despite this, it is obvious that if a sperm deficiency is linked to a genetic background, the problem may be passed to the next generation. Hence, artificial oocyte activation needs to be considered as experimental and its application requires thorough consultation with the patient. In conclusion, for patients with 0% or 1 29% fertilization in a standard ICSI cycle artificial oocyte activation may have a beneficial effect in the following treatment cycle. However, these data show that artificial oocyte activation should not be randomly applied and, especially for patients with 30 50% fertilization rates, it may not improve the overall outcome.

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Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+) release and are unable to initiate the first step of embryo development. J. Clin. Investig 118, 3671 3681. Declaration: The authors report no financial or commercial conflicts of interest. Received 12 July 2011; refereed 1 February 2012; accepted 2 February 2012.