Abstract
The refinement of gamete micromanipulation techniques has made conception possible for couples with male factor infertility who otherwise would remain childless. Moreover, intracytoplasmic sperm injection (ICSI) has ensured that such refractory cases can now generate offspring as successfully as in couples that merely require in vitro insemination. However, despite the now sterling record of ICSI it does not assure a successful outcome for every patient. This can be due, for instance, to the inability of the spermatozoon to activate the oocyte, and applies obviously in cases where spermatozoa are absent from the ejaculate or testicular biopsy. In the present paper we describe in detail the reasons for such failure and review the options that may help overcome it. In particular, we outline the treatment protocol for the situation in which spermatozoa are unable to induce oocyte activation. Further, we report on the clinical outcome achieved with spermatozoa retrieved from the testis, and in cases of extreme oligozoospermia we also explore the option of replicating a single spermatozoon while gaining information on its genomic content. For the most extreme situation in which men have no identifiable germ cells, we will discuss the current status of efforts to accomplish neo-gametogenesis through embryonic stem cell differentiation.
| Abbreviations | ||
| ICSI: | = | intracytoplasmic sperm injection |
| IVF: | = | in vitro fertilization |
| PGD: | = | preimplantation genetic diagnosis |
| PN: | = | pronuclei |
| MII: | = | metaphase II |
| PB: | = | polar bodies |
| TESE: | = | testicular sperm extraction |
| OA: | = | obstructive azoospermia |
| NOA: | = | non-obstructive azoospermia |
| PESA: | = | percutaneous epididymal sperm aspiration |
| MESA: | = | microsurgical epididymal sperm aspiration |
| ESC: | = | embryonic stem cells |
| EB: | = | embryoid bodies |
| LIF: | = | leukemia inhibiting factor |
| BMP: | = | bone morphogenic protein |
| RA: | = | retinoic acid |
| PGC: | = | primordial germ cell |
| SCP1: | = | synaptonemal complex protein 1 |
INTRODUCTION
In the course of standard in vitro fertilization (IVF), complete fertilization failure occurs in a significant proportion of cases [Barlow et al. Citation1990; Molloy et al. Citation1991; Ben-Shlomo et al. Citation1992; Lipitz et al. Citation1993; Roest et al. Citation1998; Tomas et al. Citation1998]. Although the precise reasons for such failure remain unclear, many studies point to some sperm dysfunction [Mahadevan and Trounson Citation1984; Jeulin et al. Citation1986; Liu et al. Citation1989; Liu and Baker Citation1992; Franken et al. Citation1993; Oehninger et al. Citation1997]. Among the techniques aimed at enhancing sperm-egg interaction, gamete micromanipulations immediately appeared to be the most successful. This belief brought the frenzied development of a multitude of micromanipulation methods [Gordon et al. Citation1988; Malter and Cohen Citation1989; Feichtinger et al. Citation1992; Palermo et al. Citation1992], among which the insertion of a selected spermatozoon into the ooplasm, ICSI (Intracytoplasmic Sperm Injection), emerged as the one capable of providing the most reliable fertilization [Palermo et al. Citation1992].
ICSI has been equally successful whether the sperm sample was fresh or frozen and the standard semen parameters and such other factors as the presence of anti-sperm antibodies appear to have had a negligible role in the ICSI outcome [Palermo et al. Citation1993; Citation1995; Mansour et al. Citation1995; Nagy et al. Citation1995]. Similarly, whether spermatozoa were ejaculated or were retrieved surgically from the epididymis or testis, appears to be irrelevant to the outcome [Tournaye et al. Citation1994; Palermo et al. Citation1995]. Moreover, ICSI's dependability has broadened its initial use from a technique capable of overriding the dysfunctionality of spermatozoa to one that may partly compensate for problems with the egg. Indeed, ICSI has allowed successful fertilization when only a few and/or abnormal oocytes were available [Ludwig et al. Citation1997]. Stripping cumulus cells off the oocytes allows a direct visualization of the fact that oocyte maturation occurred, therefore, offering a woman with a limited number of oocytes a much greater chance of successful fertilization. In fact, the availability of ICSI has been instrumental in some European countries e.g., Italy and Germany in circumventing restrictive legislation that limits the number of oocytes inseminated or embryos to be replaced [Ludwig et al. Citation1999; Citation2000; Benagiano and Gianaroli Citation2004].
ICSI has also made possible the consistent fertilization of cryopreserved oocytes [Porcu et al. Citation1997]. This is an advantage since freezing can lead to a premature exocytosis of cortical granules and to zona hardening that inhibits natural sperm penetration [Johnson Citation1989; Schalkoff et al. Citation1989; Vincent et al. Citation1990; Van Blerkom and Davis Citation1994]. ICSI is also the preferred conception method during the application of preimplantation genetic diagnosis (PGD). It avoids sperm DNA zona contamination and by enhancing the number of fertilizable oocytes subsequently increases the number of embryos available for screening [ESHRE Citation2007].
Finally, because a single spermatozoon is used, ICSI has allowed treatment of men who are virtually azoospermic (also defined as cryptozoospermic) [Bendikson et al. Citation2008]. Where such spermatogenic arrest occurs, this scenario has fueled attempts to inject immature spermatozoa or germ cells [Edwards et al. Citation1994; Fishel et al. Citation1995; Tesarik et al. Citation1995; Tsai et al. Citation2000]. However, when ICSI conception is accomplished, embryo implantation follows similar rules, at least in our experience, as all other assisted reproductive technologies, mainly regulated by the maternal age.
All the above-mentioned uses have contributed to the present view of ICSI as the preferred insemination method in many circumstances [Aboulghar et al. Citation1996; Fishel et al. Citation2000; Andersen et al. Citation2008], this being used in 2004 in nearly 60% of all reported ART cycles in Australia, New Zealand, Europe, and USA [Wang et al. Citation2008; Wright et al. Citation2008; de Mouzon et al. Citation2009]. Nonetheless, we would stress that ICSI does not guarantee successful fertilization for every patient, and complete fertilization failure can still occur [Flaherty et al. Citation1995; Liu et al. Citation1995a; Citation1995b]. Such failures have been reported to be between 1% [Esfandiari et al. Citation2005] and 3% [Liu et al. Citation1995; Bhattacharya et al. Citation2001] and may be explained by different factors related to oocytes and/or spermatozoa.
The current discussion attempts to bring further perspective as to the strengths and weaknesses of ICSI. For instance, it reports the clinical outcome after ICSI utilizing the dysfunctional and scarce testicular spermatozoa retrieved from men with compromised spermatogenesis. In addition, techniques aimed at supporting oocyte activation as well as their outcome are described. Finally, a cloning procedure is proposed for men with only a very limited number of retrievable sperm cells and for those with complete germ cell aplasia, the possibility of obtaining in vitro generated gametes is considered.
SPERM DYSFUNCTION
Complete fertilization failure (where all oocytes failed to display two pronuclei (PN) or displayed abnormal fertilization patterns such as 1PN and 3PN), is an endpoint that can occur in up to 3% of ICSI cycles as a result of multiple anomalies of the fertilization process [Liu et al. Citation1995a; Citation1995b; Moomjy et al. Citation1998; Ludwig et al. Citation1999; Bhattacharya et al. Citation2001].
In over a decade of experience at Cornell, the average number of oocytes retrieved was 10.8 per cycle (n=169,040), of which 131,751 were at metaphase II. Of the 130,658 oocytes injected, 94.4% (n=97,208) survived and 74.4% developed two pronuclei (PN). During this period, an overall 2.1% of ICSI cycles failed to be fertilized (; ).
Figure 1 Fertilization failure profile at Cornell University. Cases with complete fertilization failure are those where all oocytes failed to display two pronuclei (PN) or displayed abnormal fertilization patterns such as 1PN and 3PN.
Table 1 Patient Characteristics with Cycles of Complete Fertilization.
The principal cause of fertilization failure after ICSI has been attributed to an absence of ooplasmic activation observed in 40–70% of the injected oocytes [Sousa and Tesarik Citation1994; Flaherty et al. Citation1995; Yanagida Citation2004], a proportion of which may reflect an inability of the spermatozoon to provide an activating factor [Palermo et al. Citation1996]. Such a sperm-associated factor is considered to be responsible for triggering oocyte activation [Dozortsev et al. Citation1995a; Citation1995b; Citation1997; Parrington et al. Citation1996; Young et al. Citation2009], an event which may also be stimulated by the priming induced by the microtool during oolemma penetration [Winston et al. Citation1991; De Sutter et al. Citation1992].
A ‘living’ spermatozoon facilitates the triggering of oocyte activation and induction of normal fertilization [Dozortsev et al. Citation1995b] defined as the appearance of a zygote, which is the presence of two distinct pronuclei and two polar bodies. Thus, sperm membrane integrity tests have indicated that ejaculated immotile but viable spermatozoa [Verheyen et al. Citation1997] are capable of fertilizing an oocyte [Moomjy et al. Citation1998], as do immotile spermatozoa obtained directly from seminiferous tubules [Devroey et al. Citation1995].
The developmental competence that is acquired by the oocyte as a result of the completion of meiosis to metaphase II (MII) plays a critical role during fertilization and subsequent stages of preimplantation embryonic development [Albertini et al. Citation2003]. Several factors may be related to an inability of the oocyte to activate. First, the timing of maturational events appears to be tightly regulated [Eppig et al. Citation1996; Moor et al. Citation1998; Trounson et al. Citation2001; Albertini et al. Citation2003] and the competence to undergo nuclear and cytoplasmic maturation is acquired independently during folliculogenesis [Eppig et al. Citation1994]. Oocytes are retrieved from varying follicle sizes and they may carry an immature cytoplasm despite the premature extrusion of the first polar body. It has been postulated that such an asynchrony between nuclear and cytoplasmic maturation may be responsible for the inability of the ooplasm to support decondensation of the sperm nucleus [Sundstrom and Nilsson Citation1989; Eppig et al. Citation1994]. In support of this idea, meiotic spindle visualization reveals that these apparently mature oocytes (first polar body extruded) may still be at telophase I or prometaphase II [De Santis et al. Citation2005; Rienzi et al. Citation2005; Montag et al. Citation2006] and deemed ready for ICSI. This situation is confirmed by the presence of a meiotic spindle bridge between ooplasm and first polar body (PB).
Sperm-oolemma fusion is bypassed by ICSI, thus, allowing round-headed acrosomeless spermatozoa to fertilize. Even if these spermatozoa are characterized by structural and chromatin compaction defects ( and ) [Liu et al. Citation1995a; Citation1995b], attempts can be made to enhance the chance of fertilization by including oocyte activating factors as the sperm is injected [Palermo et al. Citation1997a; Morozumi et al. Citation2006]. Oocyte activation agents include calcium ionophore [Hoshi et al. Citation1992], electrostimulation [Yanagida et al. Citation1999], and strontium [Yanagida et al. Citation2006]. Such adjunct treatments help to activate the oocyte by increasing the Ca2+ permeability of the cell membrane, thereby letting extracellular Ca2+ flow into the cell and induce release of Ca2+ from the intracellular calcium storage. In one of the first series of assisted oocyte activations performed in patients with previous cycles of failed fertilization [Heindryckx et al. Citation2005], the authors first injected a spermatozoon together with CaCl2 and thereafter, a calcium ionophore; this dual exposure resulted in an overall fertilization of over 70%.
Figure 2 (A) Sperm DNA fragmentation assessed by sperm chromatin dispersion test. (B) Sperm DNA fragmentation assessed by TUNEL assay.
In a prospective at our center (IRB 071200953), four couples with a history of fertilization failure agreed after careful and appropriate counseling to an experimental treatment in which their spermatozoa were treated prior to injection with streptolysin O (30 min), and after injection oocytes were exposed to Ca2+ ionophore to promote oocyte activation. Fertilization was assessed and clinical pregnancies were judged according to the presence of at least one fetal heartbeat (FHB). The patients (maternal age 36.4±2) in the study had an average of 2.2 prior cycles with no fertilization. The sperm parameters had a concentration of 58.6±40×106/ml with a mild impairment of motility (24.5±10%) and morphology (2.3±1%). The cumulative prior cycles (n=9) yielded an average of 9.8 oocytes, all failing to fertilize. The study cycles (n=5) produced an average of 12.6 oocytes with 42.1% being fertilized—a remarkable improvement. After activation treatment, 5 of the 38 oocytes degenerated. The cleavage rate of the fertilized oocytes was 87.5% with the mean number of blastomeres being 7.2±1 on day 3, and a mean fragmentation rate of 9.5%±3. Transfers were performed in four cycles with a mean number of 3.5 embryos transferred per procedure. Two cycles tested positive for βhCG with one ongoing pregnancy. These preliminary results suggest that couples with fertilization failure can be ‘rescued’ by spermatozoa pre-treatment combined with post-injection oocyte activation.
DYSSPERMATOGENESIS
The fact that ICSI provides a way of achieving fertilization regardless of the characteristics of the spermatozoon has justified the application of this approach to crypto- and even azoospermic patients. Characterized by fluctuations in the effectiveness of the germ cell to progress through the meiotic line, this pattern leads to transient azoospermia, rendering unpredictable the identification of spermatozoa in the ejaculate. In such cases, a meticulous extended sperm search protocol [Ron-El et al. Citation1997; Strassburger et al. Citation2000; Bendikson et al. Citation2006; Citation2008] can sometimes yield a number of spermatozoa sufficient to inseminate the entire cohort of oocytes. When no spermatozoa are found, recovery from the testis is an option that, besides involving some iatrogenic risks, can still fail to yield any spermatozoa. In the management of cryptozoospermic patients, a strategy that involves the collection of multiple frozen specimens can help to ensure the availability of male gametes at the time of ICSI [Koscinski et al. Citation2007].
When infertile couples present for consultation a routine semen analysis is requested and in about 3% of the cases no spermatozoa are identified and require centrifugation of the ejaculate at 3,000×g. At our center, 146 cases identified as having no spermatozoa in their initial semen analysis, centrifugation resulted in a concentration of 26,000/ml with an average motility of 21.7%±23. Of the oocytes injected, a fertilization rate of 62.0% was achieved with sperm recovered in this way, resulting in a 37.0% (54/146) clinical pregnancy rate. Some of these patients were counseled by a urologist for an eventual testicular sampling back-up in case no spermatozoa were found on the day of the retrieval. Conversely, men with secretory azoospermia scheduled for TESE (testicular sperm extraction) had their ejaculate screened on the day of surgery for eventual presence of spermatozoa. If an adequate number of spermatozoa were identified, the TESE procedure was cancelled.
Definitive azoospermia represents the most extreme form of male infertility and it affects approximately 10 to 15% of all infertile couples screened [Willott Citation1982; Jarow et al. Citation1989]. It can be classified as obstructive azoospermia (OA), as in the congenital bilateral absence of the vas deferens or epididymal blockage, or non-obstructive azoospermia (NOA) as a result of testicular failure [Reijo et al. Citation1996; Girardi et al. Citation1997]. In men with OA, spermatozoa can be retrieved in most cases [Tournaye et al. Citation1997] by percutaneous (PESA) or microsurgical (MESA) epididymal sperm aspiration. More invasive techniques such as fine needle aspiration of the testicle are used when the epididymal approach is not feasible. In NOA, seminiferous tubule sampling is the most reliable method for sperm retrieval. Due to the scattered presence of spermatogenic foci, a microsurgical sampling of engorged seminiferous tubules present a greater chance to find spermatozoa [Schlegel et al. Citation1997]. Spermatozoa can be retrieved in about 50–70% of non-obstructive azoospermic men with a spermatogenic defect [Palermo et al. Citation1999; Tournaye et al. Citation2002; Schlegel Citation2006], but at times this approach requires an extensive and tedious search [Bendikson et al. Citation2006; Citation2008; Chen et al. Citation2009]. In a small cohort of couples (n=461) who underwent TESE at our facility, a total of 632 ICSI cycles were carried out. Of those, 535 had a sperm search that lasted less than one hour, while the study group (n=97) had an extended sperm search that ranged from 61 to 390 min. It appeared that the use of testicular spermatozoa recovered following such extended searches impaired fertilization (55.6% vs. 41.7%; P=0.0001, respectively) and delivery rates (40.2% vs. 24.2%; P=0.007, respectively) particularly so when the sperm search lasted over 3 h [Bendikson et al. Citation2006; Citation2008]. Thus, for these patients at least ICSI provides a real chance of conception even if pregnancy rates are lower [Vernaeve et al. Citation2003; Nicopoullos et al. Citation2004].
In the last fourteen years, 16,980 ICSI cycles from all semen sources have been performed at Cornell. A total of 143,127 oocytes were injected, with a fertilization rate of 73.6%. When the outcome was compared according to semen origin, the ejaculated group yielded a higher fertilization rate than those surgically retrieved (P=0.0001; ). When the clinical pregnancies were compared, however, ejaculated cycles had a comparable outcome (39.8%) to the testicular group (41.4%) and the epididymal group had the most successful outcome (53.7%). This indicates that a maternal age factor can compensate for the defective sperm production, however, among azoospermic men, obstructive is confirmed to be superior.
Figure 3 Fertilization and pregnancy rates according to semen origin.
The therapeutic possibilities of ICSI go further as even spermatid conception was proposed for the treatment of azoospermia [Edwards et al. Citation1994] and the gametes at the elongated or at the round spermatid stage were used to achieve fertilization [Fishel et al. Citation1995; Citation1996; Tesarik Citation1996; Yamanaka et al. Citation1997; Sofikitis et al. Citation1998a;. Citation1998b]. Although pregnancies with the pre-spermiogenic stage were reported and even less verisimilarly from primary spermatocytes [Tesarik and Greco Citation1999], these techniques failed to gain popularity and instead fuelled a controversy as to their usefulness and safety [Tesarik et al. Citation1995; Sousa et al. Citation1999]. Whenever round spermatids are identifiable in a testicular biopsy specimen, elongated spermatids and immature spermatozoa are also present [Silber and Johnson Citation1998]. In fact, spermatids may lead to impaired oocyte activation and have adverse effects on embryo development [Yanagimachi Citation2005]. In addition, these pre-spermiogenic stages may lack a fully developed centrosome [Schatten Citation1994; Palermo et al. Citation1997b; Schatten and Sun Citation2009] and suffer from incomplete genomic imprinting [Palermo et al. Citation2008].
As a practical matter, clearly the successful treatment of cryptozoospermic and azoospermic men depends on the ability to isolate haploid cells from a testicular biopsy. Although currently experimental, haploid germ cells may be enriched by flow cytometry after seminiferous tubule digestion and these post-meiotic cells may be retrieved by fluorescence activated cell sorting [Akerman et al. Citation2000].
VIRTUAL AND DEFINITIVE AZOOSPERMIA
Clearly, the difficulty in retrieving spermatozoa represents the main hindrance in the treatment of men with extremely compromised spermatogenesis. Therefore, the possibility to replicate a spermatozoon for insemination would represent a solution to this problem. The original idea for this stems from the need to obtain genetic information on a specific gamete that can then itself be used for ICSI. Although it is possible to routinely assess the chromosomal complement of individual spermatozoa () [Palermo et al. Citation2002], those cells cannot be used for injections. It has been proposed, however, that replication of the male gamete through a host ooplasm may indeed provide this option [Willadsen et al. Citation2000]; and a test run for this approach was conducted even on human gametes [Kuznyetsov et al. Citation2007].
Figure 4 Cytogenetic assessment on spermatozoa by fluorescent in situ hybridization.
We pursued the idea of sperm replication further by attempting to generate mouse conceptuses capable of undergoing full term development to enhance a spermatozoon's reproductive power () [Takeuchi et al. Citation2008]. As previously shown, enucleated metaphase II (MII) oocytes injected with single spermatozoon underwent embryo-like cleavage as haploid androgenotes with a high efficiency [Palermo et al. Citation2009]. Of 91 intact MII oocytes, 95.6% survived enucleation and following single sperm injection displayed a single male pronucleus. All of these 87 androgenones developed to the two cell, 75 (82.4%) to the four cell, and 50 (64.8%) to morula stage, but only 3 (3.3%) developed a blastocoel. A total of 42 karyoplasts isolated from 13 quartets were fused to generate 41 (97.6%) zygotes that developed to 33 (80.5%) blastocysts (). Transfer of 30 blastocysts into the uterine horns of pseudopregnant females yielded 12 offspring (40.0%). Such pseudoblastomeres generated by the replication of a spermatozoon maintained their haploid status, and participated in syngamy and supported embryo development. This procedure appears reliable and not plagued by the epigenetic defects typical of cloning.
Figure 5 Male genome cloning. (A) To generate a haploid androgenote, a metaphase II oocyte will be enucleated and injected with a single spermatozoon. A haploid andrognote displaying one pronucleus will be cultured to allow embryo-like cleavage. Each blastomere maintains its original ploidy. (B) Karyoplasts will be isolated from blastomeres of haploid androgenotes, while haploid parthenotes will be generated by exposing intact metaphase II oocytes to SrCl2. A biparental zygote will be reconstituted by electrofusing an androgenetic karyoplast with a parthenote, and cultured up to the blastocyst stage followed by transferring to a pseudo-pregnant female.
An alternative approach to provide a definitive treatment for azoospermic men could involve the generation of male gametes through differentiation of embryonic stem cells (ESC). Recent studies have demonstrated that ESC can indeed differentiate into germ cells and even into post-meiotic stages [Hubner et al. Citation2003; Toyooka et al. Citation2003; Geijsen et al. Citation2004; Nayernia et al. Citation2006]. Although the production of oocyte-like germ cells appears to be optimal in a bidimensional environment, the differentiation of the male germ cell seems to benefit from a tridimensional structure as within embryoid bodies (EB).
Developing EBs in standard DMEM medium devoid of leukemia inhibiting factor (LIF), yield germ cells only at a rate of about 1%. Growth factors such as LIF and the bone morphogenic protein (BMP) derivatives [Clark et al. Citation2004] are considered essential to support germ cell proliferation, self-renewal, and sustain their survival, while other factors such as retinoic acid (RA) would push them to enter meiosis [Geijsen et al. Citation2004; Nayernia et al. Citation2006]. By modulating the presence of these factors (BMP4, 7, and 8b; 1 μM or 10 μM RA) along with EB formation, we attempted to recreate a seminiferous tubule-like environment [Neri et al. Citation2006; Palermo et al. Citation2009] while enhancing primordial germ cell (PGC) proliferation [(Silva et al. Citation2009] and their progression towards post-meiotic stages. Quantitative PCR assessment indeed confirmed the progressive loss of stemness together with the appearance of markers specific for post-migratory PGCs and spermatogonia stem cells. The subtle presence of post-meiotic stages was confirmed by the expression of synaptonemal complex protein 1 (SCP1) and Tekt1, indicating their progression through the spermatogenic maturation line with possible appearance of spermatids. Our findings have been confirmed by the report of mouse offspring following fertilization with sperm-like cells derived in a culture environment. However, significant concerns remain about the functionality of these cells and the ability of the genome to develop a proper imprinting pattern, as indicated by the phenotypic abnormalities of the short-lived offspring [Nayernia et al. Citation2006]. Nonetheless, it is at least a possibility worth pursuing that, in spite of these difficulties, the generation of gametes from ESCs might well represent a therapeutic option for men with germ cell aplasia.
CONCLUSIONS
The introduction of ICSI represents a considerable milestone in the treatment of male factor infertility. The efficiency of the technique has resulted in its general popularity and has made ICSI the preferable way to fertilize an oocyte. In fact, ICSI can be carried out with spermatozoa that are dysmorphic, dysfunctional, immature, or coated with anti-sperm antibodies. However, a proportion of couples fail to achieve fertilization with this technique at times due to ooplasmic problems or because their spermatozoa lack the ability to activate oocytes. Modification of the injection protocol by including agents that permeabilize the spermatozoa to induce intracellular calcium release can obviate this failure. Azoospermic men with compromised spermatogenesis can successfully be treated as long as a few developed spermatozoa can be retrieved by semen centrifugation or surgically. While utilization of spermatids and more immature forms of the male gamete has proven unreliable (very inconsistent), replication techniques have produced individual spermatozoa capable of generating multiple mouse offspring while providing insight into the genome of the spermatozoon selected. For men with complete germ cell aplasia, some hope resides in the differentiation of embryonic stem cells into germ cells, and hopefully, the in vitro generation from these of functional gametes.
ACKNOWLEDGMENTS
We thank the clinicians, scientists, embryologists, and nursing staff of The Center for Reproductive Medicine and Infertility. We are most grateful to Professor J. Michael Bedford for editing the manuscript, Eugene Ermolovich, Christopher Chen, Zahraa Kollmann, and Miriam Feliciano for assisting with data collection. QVN was funded by the grant ULI RR024996 of the Clinical and Translational Science Center at Weill Cornell Medical College.
Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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