Application of a ready-to-use calcium ionophore increases rates of fertilization and pregnancy in severe male factor infertility

Application of a ready-to-use calcium ionophore increases rates of fertilization and pregnancy in severe male factor infertility Thomas Ebner, Ph.D., a Maria K oster, Ph.D., b Omar Shebl, M.D., a Marianne Moser, Ph.D., a Hans Van der Ven, M.D., b Gernot Tews, M.D., a and Markus Montag, Ph.D. b,c a Landes- Frauen- und Kinderklinik, Kinderwunschzentrum, Linz, Austria; b Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Bonn; and c Gynecological Endocrinology and Fertility Disorders, University of Heidelberg, Heidelberg, Germany Objective: To analyze whether a ready-to-use calcium ionophore improves outcomes, from fertilization to live birth, in patients with severe male factor infertility. Design: Artificial oocyte activation offered to applicable patients over a 20-month period. Setting: Specialized in vitro fertilization (IVF) centers in Austria and Germany. Patient(s): Twenty-nine azoospermic and 37 cryptozoospermic men. Intervention(s): Mature oocytes treated with a ready-to-use Ca 2þ -ionophore (GM508 Cult-Active) immediately after intracytoplasmic sperm injection (ICSI). Main Outcome Measure(s): Rates of fertilization, implantation, clinical pregnancy, and live birth. Result(s): Patients had had 88 previous cycles without artificial activation that resulted in a fertilization rate of 34.7%, 79 transfers (89.8%), and 5 pregnancies, which all spontaneously aborted except one. After artificial oocyte activation, the fertilization rate was 56.9%. In terms of fertilization rate, both azoospermic (64.4%) and cryptozoospermic (48.4%) men statistically significantly benefited from use of the ionophore. In 73 transfer cycles, positive b-human chorionic gonadotropin levels were observed in 34 cases (46.6%) and 29 cycles (39.7%) that ended with a clinical pregnancy. The corresponding implantation rate was 33.3%. Four spontaneous abortions occurred (11.8%), and 32 healthy children were born. Conclusion(s): This is the first prospective multicenter study on artificial oocyte activation in severe male factor infertility. Present data indicate that a ready-to-use calcium ionophore can yield high fertilization and pregnancy rates for this particular subgroup. In addition to fertilization failure after ICSI, severe male factor infertility is an additional area for application of artificial oocyte activation. (Fertil Steril Ò 2012;98:1432 7. Ó2012 by American Society for Reproductive Medicine.) Key Words: Azoospermia, Ca 2þ -ionophore, cryptozoospermia, TESE Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/ebnert-calcium-ionophore-severe-male-factor-infertility/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. In vivo or in conventional in vitro fertilization (IVF), the fertilization process may be subdivided into six major events: [1] cumulus cell penetration, [2] sperm-oocyte binding and penetration, [3] sperm-oocyte fusion, Received May 21, 2012; revised June 27, 2012; accepted July 23, 2012; published online August 24, 2012. T.E. is a consultant for Gynemed, and has received payment for expert testimony for MSD and development of educational presentations by University for Life, Graz, Austria. M.K. has nothing to disclose. O.S. has nothing to disclose. M.M. has nothing to disclose. H.V.d.V. has nothing to disclose. G.T. has nothing to disclose. M.M. is a consultant for Unisense Fertilitech and ilabcomm GmbH Consulting Company, and has received payment for lectures from Merck Serono, Ferring, and MSD, and payment for development of educational presentations by Uniforlife, Graz, Austria. Reprint requests: Thomas Ebner, Ph.D., Landes- Frauen- und Kinderklinik Linz, Kinderwunsch Zentrum, Krankenhausstr. 26 30, A-4020 Linz, Upper Austria, Austria (E-mail: thomas.ebner@ gespag.at). Fertility and Sterility Vol. 98, No. 6, December 2012 0015-0282/$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.07.1134 [4] oocyte activation, [5] sperm processing, and [6] pronucleus formation (1). The pre-sperm-penetration phase (events 1 to 3) may be bypassed using common micromanipulation techniques such as intracytoplasmic sperm injection (ICSI). Consequently, the number of fertilization failures is reduced in ICSI patients as compared with IVF patients. The complexity of the fertilization process may explain its susceptibility to disturbances potentially leading to impaired fertilization rates in spite of the presence of a presumably normal 1432 VOL. 98 NO. 6 / DECEMBER 2012

Fertility and Sterility spermatozoon (2, 3). The frequency of total fertilization failures in ICSI cycles was reported to be approximately 1% to 3% (4, 5), most of these being the result of impaired semen characteristics or a very low number of oocytes collected. In such cases, repeated ICSI proved useful (4, 5); however, some patients will have to face repeated fertilization failure in spite of normal sperm parameters and good ovarian response (5, 6). Both electrical oocyte activation (7 9) and modified ICSI techniques (6, 10) have been suggested for rescuing such cycles. Because such methods are more invasive than conventional micromanipulation techniques, general acceptance in the field of assisted reproduction technology (ART) is still lacking. Rather, in cases of reduced or failed fertilization after ICSI, artificial oocyte activation is induced with a variety of chemical agents such as 6- dimethylaminopurine or strontium chloride, to name but a few (11). Of these artificial activating substances for human oocytes, calcium (Ca 2þ ) ionophores such as ionomycin, calcimycin (A23187), or a combination of two ionophores are the most popular (12 16). From the beginning (17), both types of Ca 2þ ionophores have been successfully used in cases of complete globozoospermia (18 20) and previous (repeated) fertilization failure after ICSI (21 24). In the literature, these case reports were followed by larger case series (25 27) showing that both ionomycin and calcimycin were indeed feasible options in cases of previous fertilization problems. To date, the largest (retrospective) study using a compounded A23187-solution was published by Montag et al. (28) indicating that fertilization rates close to 50% can be expected in ICSI cases previously facing fertilization problems. In-house mixing of ionophore solutions is a technical challenge because A23187 is not soluble in water. Furthermore, in times of the implementation of the European Tissue Directive, compounding of agents could be a potential source of contamination. Montag et al. (29) were the first to prospectively apply a commercially available ready-to-use solution of A23187 in a larger series of patients with total or major fertilization failure after ICSI (based on the absence of two pronuclei). Our prospective study evaluated a potentially positive influence of a ready-to-use Ca 2þ ionophore on patients with severe male factor infertility such as cryptozoospermia or azoospermia. MATERIALS AND METHODS This prospective multicenter analysis was performed between August 2009 and March 2011 (20 months) at two IVF clinics in Austria (Kinderwunsch Zentrum Linz) and Germany (University of Bonn). All patients involved gave written consent, and institutional review board approval was granted (reference number 4-J-09). Because of our practical experience with the ready-to-use calcium ionophore in cases of severe male infertility (29) and the history of the patients involved, splitting of oocytes (with and without A23187) was avoided, and the ionophore treatment was fully relied on. We considered the previous cycles to be the ideal control group because the cycle characteristics and laboratory variables had not changed noticeably: that is, the culture conditions, testicular sperm extraction (TESE) material, embryologists performing the ICSI, and embryo transfer procedures were similar. According to our inclusion criteria only patients with azoospermia (44%) or cryptozoospermia (56%) (e.g., showing <0.1 spermatozoa/ml) were asked to participate. Figure 1 illustrates that 78% of all potential candidates could be included. These 66 couples previously had 88 cycles with only a single live birth. The number of previous cycles was identical at both clinics (1.3 to 1.4). In the present prospective trial, the majority of female partners (32.1 4.8 years) were stimulated according to a long protocol; however, 15.4% of the patients had an antagonist protocol. If the former regimen was chosen, downregulation was required before stimulation started with recombinant gonadotropins. In the antagonist protocol, the same FSH products were started on day 3 of the cycle. In addition, a gonadotropin-releasing hormone (GnRH) antagonist was administered after 5 to 6 days of stimulation, depending on the size of the leading follicles. In all cycles, ovulation was induced with 10,000 IU human chorionic gonadotropin (hcg). Oocyte retrieval was performed transvaginally 36 hours after hcg administration. It is important to note that the FIGURE 1 Azoospermia (43 cycles) Potentially relevant and screened patients with severe male factor (n=85) Patients included who gave written consent (n=66) ICSI ready-to-use ionophore Cryptozoospermia (32 cycles) In vitro culture to day 3 (n=55) or 5 (n=20) Embryo or blastocyst transfer (n=73) Outcome Refused to participate (n=7) Insufficient # oocytes (n=12) Cycle cancellation (n=2) Application of a ready-to-use calcium ionophore in severe male factor infertility: schematic presentation of the study design and outcome. Ebner. Ionophore and severe male infertility. Fertil Steril 2012. VOL. 98 NO. 6 / DECEMBER 2012 1433

ORIGINAL ARTICLE: ANDROLOGY stimulation regimens were not changed after the previous cycles because regular fertilization was achieved; thus, any theoretical improvement in outcome should be most likely related to the usage of A23187. In cases of cryptozoospermia, raw ejaculate was processed with density gradient (45% and 90%) or mini-swimup techniques to accumulate spermatozoa for ICSI. All azoospermic patients had TESE material cryopreserved. In short, TESE began with a small incision being made in the scrotal skin and carried through the peritoneal tunica vaginalis. Small pieces of testicular tissue were extruded through an opening in the tunica albuginea. The cutoff samples were placed in tubes containing culture medium for additional manipulation. Biopsy samples were mechanically disaggregated within 30 minutes under sterile conditions in a Petri dish. As many spermatozoa as possible were accumulated in the liquid supernatant. After loading, the cryostraws were frozen by an automatic slow-freezing procedure. Thawing was performed as previously published elsewhere (30, 31). Regardless of the sperm source used, all injected oocytes were incubated for 15 minutes in 200 ml of a ready-to-use A23187 solution (28) called GM508 Cult-Active (Gynemed) immediately after ICSI. After three washing steps in 500 ml of culture medium each, injected eggs were transferred to microdroplets (30 ml) of sequential culture media until the fertilization check, which was performed 16 to 20 hours after ICSI. Regular fertilization was assumed if two pronuclei as well as two polar bodies were identified. On day 2 of preimplantation development, all embryos were scored according to the number and size of the blastomeres as well as the presence of fragmentation and multinucleation. For the day-3 assessment, the same morphologic parameters were used (32). It should be noted that legal restrictions in Germany required that blastocyst culture could only be performed in the Kinderwunsch Zentrum Linz, Austria. In these cases, the blastocysts were scored according to their expansion, and the quality of the inner cell mass and trophectoderm, respectively. Surplus zygotes (University of Bonn) and blastocysts were cryopreserved. Although the average number of embryos transferred differed due to the legal background in both countries, the difference (1.4 in Linz vs. 1.7 in Bonn) did not reach statistical significance. Because the implantation, pregnancy, and livebirth rates did not differ between the patients with azoospermia or cryptozoospermia and between the clinics, the data were pooled. Differences between continuous variables were assessed with a t-test for independent samples and with a chi-square test for categorical variables. P<.05 was considered statistically significant. RESULTS In the present prospective approach, oocyte collection in 66 patients (75 cycles) resulted in 1,625 cumulus-oocyte complexes. After denudation, it turned out that only 1,370 (84.3%) of 1,625 in fact were mature and, consequently, available for ICSI. As indicated in Table 1, an overall fertilization rate of almost 57% could be achieved using the ready-to-use calcium ionophore, which was a statistically significant increase as compared with the previous cycles of the same couples (P<.001). On a per patient basis, 50 (75.8%) of 66 patients benefited from the ionophore application. The azoospermic patients had statistically significantly higher fertilization rates than the cryptozoospermic ones. This had also been true for the previous cycles (P¼.012), but was even more evident in this study (P<.001). Embryo quality was not statistically significantly different when comparing the present and previous treatments. Embryo quality was found to be similar in the azoospermic and cryptozoospermic patients. Although blastocyst culture was only done in one IVF center and thus the sample numbers were reduced, blastocyst formation differed in highly statistically significant manner (P<.001). Pregnancy rates (positive b-hcg levels) with blastocyst transfer were slightly higher (53.9%) as compared with day-3 transfers (42.6%), but the difference was not statistically significant (P¼.35). As a general trend, fewer patients had their embryo transfer canceled as compared with their previous treatments (P¼.06). Ultimately, the rates of positive b-hcg levels, implantation, and clinical pregnancy were statistically significantly improved compared with the previous trials (P<.001). No difference in these rates was observed with respect to the type of male infertility. Consequently, the live-birth rate was found to be statistically significantly increased as well (P<.001). Specifically, 20 singleton and 6 twin pregnancies were finally delivered, resulting in 32 healthy children. No malformations were observed. DISCUSSION Although the fertilization rate with ICSI is considered to be the highest among all available assisted reproduction techniques, TABLE 1 Results of the prospective application of A23187 as compared with the previous cycles without the use of ionophore. Previous A23187 cycle cycles P value No. of cycles 75 88 COC collected 9.6 5.8 11.2 8.3 NS (mean SD) Fertilization rate 379/666 (56.9) 244/704 (34.7) <.001 Azoospermia 228/354 (64.4) b 91/220 (41.4) c <.001 Cryptozoospermia 151/312 (48.4) b 153/484 (31.6) c <.001 Blastocyst formation a 67/119 (56.3) 20/79 (25.3) <.001 No. of ETs 73 (97.3) 79 (89.8) NS Implantation rate 42/126 (33.3) 6/150 (4.0) <.001 Positive b-hcg 34 (46.6) 6 (7.6) <.001 Clinical pregnancy rate 29 (29.7) 5 (6.3) <.001 Live-birth rate 25 (34.2) 1 (1.3) <.001 Children born 32 1 Note: Values in parentheses are percentages. b-hcg ¼ b human chorionic gonadotropin; COC ¼ cumulus-oocyte complex; ET ¼ embryo transfer; NS ¼ not statistically significant; SD ¼ standard deviation. a Exclusive data from Kinderwunsch Zentrum Linz. b P<.001. c P<.05. Ebner. Ionophore and severe male infertility. Fertil Steril 2012. 1434 VOL. 98 NO. 6 / DECEMBER 2012

Fertility and Sterility complete fertilization failure is a threat (5, 33), especially when nonejaculated spermatozoa must be used (34, 35). Ultrastructural analysis of fertilization failure after ICSI has revealed that a deficiency in the mechanism of oocyte activation is the most common cause (2, 36). Recent data have linked these cases with oocyte activation problems due to aberrant expression, localization, and/or protein structure of phospholipase C zeta (PLCz) (37, 38). The discovery of this soluble sperm-derived phospholipase with distinctive properties was a breakthrough (39). Its presence in sperm correlates with the potential to induce Ca 2þ oscillations in the oocyte (via the inositol triphosphate pathway), which is a prerequisite for proper oocyte activation (40, 41). In the male germ line, PLCz is expressed from spermatids to spermatozoa but not in the earlier steps of spermatogenesis (39), clearly indicating that this phospholipase is expressed in testicular spermatozoa with all resultant consequences if the biological function is somewhat aberrant. There are numerous approaches to overcoming the problem of activation/fertilization failure after ICSI (11). Among others, application of calcium ionophores has proved particularly advantageous because of their ease of implementation. As exposure of human gametes to calcium ionophores has not yet been found to be associated with evidence of toxicity or detrimental outcome, its widespread use in cases of previous fertilization failure has become a routine technique. However, these optimistic results should not disguise that any calcium ionophore is an artificial activation agent that causes a singular prolonged Ca 2þ rise while failing to elicit physiologic patterns of Ca 2þ release. Thus, its application should be limited to special indications. It is known that the ICSI method per se does influence expression of numerous genes in mouse (42) and bovine (43) models. Indeed, it has been suggested that a different pattern of calcium oscillation after ICSI (44) may trigger a different wave of gene activation (42). This might also hold true for artificial oocyte activation; using strontium chloride to activate eggs leads to gene activation differences as compared with conventional IVF and ICSI (43). To date, no data on A23187 and gene expression are available. However, studies on artificial oocyte activation in cases of severe male infertility (apart from globozoospermia) have been scarce. Moaz et al. (25) reported a significant increase of fertilization rates in patients with extreme forms of isolated teratozoospermia. In their study, ionomycin helped to overcome fertilization problems in spermatozoa with amorphous and tapered heads but not in those with bent necks. Nasr-Esfahani et al. (45) confirmed the rationality of using artificial oocyte activation in cases of severe teratozoospermia and azoospermia. Although ionophores are not always successful (46), deliveries even in the presence of nonviable testicular spermatozoa are conceivable after treatment with A23187 (47). Borges et al. (26, 27) also evaluated the possible influence of artificial oocyte activation in patients with surgical sperm retrieval. In younger patients (<36 years of age), those with ejaculated and epididymal spermatozoa benefited most from the application of A23187. In the latter cohort, embryo quality statistically significantly improved (26). In a subsequent analysis (27), the investigators examined whether the type of azoospermia might explain the observed absence of any positive effect of A23187 (26) in TESE patients; however, neither obstructive nor nonobstructive TESE patients showed better rates of fertilization, implantation, and pregnancy, when A23187 was used. Their study is of particular interest because our results strongly suggest that even azoospermic patients could successfully be treated with ionophores. In our data set, azoospermic men (but also cryprozoospermic men) benefited tremendously (P<.001) with respect to the rates of fertilization, implantation, clinical pregnancy, and live birth. Several reasons may potentially explain this discrepancy. The ready-to-use agent GM508 Cult-Active, which is produced under highly standardized and sterile conditions, may be more effective than a compounded solution (26, 27). The wide difference in dosage is a much more likely reason. Theoretically, immature testicular spermatozoa need higher levels of Ca 2þ ionophore than do spermatozoa from other sources, such as ejaculated or epididymal sperm. Our evaluation used a commercially available product (Cult- Active) with a concentration of A23187 that was more than three times higher than previously used in the literature (26, 27), which could have led to the observed benefits in TESE patients. Another possible reason could be a greater variance in the TESE patient population. There is evidence from the literature that ICSI with testicular spermatozoa is less successful in men with nonobstructive azoospermia than in men with obstructive azoospermia (48). More than three quarters of the men in our study suffered from obstructive azoospermia, and thus our cohort may have begun with a better prognosis. The finding that cryptozoospermic patients have worse outcomes than TESE patients is unsurprising. It has been suggested that posttesticular sperm DNA fragmentation is even more frequent because testicular spermatozoa show lower levels of DNA damage than epididymal or ejaculated sperm (49, 50). 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