Human Reproduction Vol.16, No.12 pp. 2640–2645, 2001
Assisted reproduction with in-vitro-cultured testicular spermatozoa in cases of severe germ cell apoptosis: a pilot study Jan Tesarik1,2,5, Ermanno Greco3 and Carmen Mendoza2,4 1Laboratoire 3European 5To
d’Eylau, Paris, France, 2MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada, Spain, Hospital, Via Portuense 700, Rome, Italy, and 4University of Granada, Campus Fuentenueva, 18004 Granada, Spain
whom correspondence should be addressed at: 55 rue Saint-Didier, 75116 Paris, France. E-mail:
[email protected]
BACKGROUND: Apoptosis-related cell damage is known to compromise success rates of assisted reproduction with ejaculated spermatozoa. This study was undertaken to determine whether the frequency of apoptosis-related cell damage and reproductive performance of testicular spermatozoa from men with non-obstructive azoospermia can be improved by in-vitro culture. METHODS: Testicular tissue samples were cultured for 2 days in the presence of 50 IU/l FSH and 1 µmol/l testosterone. The frequency of spermatozoa showing DNA strand breakage and plasma membrane phosphatidylserine externalization was compared in before-culture and after-culture samples. The afterculture samples were used in assisted reproduction attempts. RESULTS: In a group of 11 azoospermic patients with at least two previous intracytoplasmic sperm injection (ICSI) failures, the incidence of DNA strand breakage was high in living testicular spermatozoa from before-culture samples, but significantly lower in after-culture samples (96 versus 30%, P < 0.001). The same applied to the incidence of phosphatidylserine externalization in the motile sperm subpopulation from the before-culture and after-culture samples (83 versus 6%, P < 0.001). Seven ongoing clinical pregnancies (six with fresh embryos and one with cryopreserved embryos) were established. CONCLUSIONS: Severe testicular sperm apoptosis may become a new indication for testicular tissue in-vitro culture before ICSI. Key words: apoptosis/assisted reproduction/in-vitro culture/non-obstructive azoospermia/testicular spermatozoa
Introduction Early studies comparing the success rates of intracytoplasmic sperm injection (ICSI) in different clinical indications reported a surprising lack of dependence of ICSI results on a variety of sperm parameters including the source (ejaculate, epididymis or testis) from which the spermatozoa used for ICSI were obtained (Nagy et al., 1998). However, some studies pointed out certain pathological conditions leading to lower ICSI success rates. For instance, several recent reports show that the use of testicular spermatozoa from patients with nonobstructive azoospermia leads to lower fertilization and implantation rates as compared with the use of ejaculated or epididymal spermatozoa (Palermo et al., 1999; Ubaldi et al., 1999; Balaban et al., 2001), to a decrease in the proportion of embryos that progress to the blastocyst stage (Balaban et al., 2001) and to unusually high spontaneous abortion rates (Ghazzawi et al., 1998). These observations are consistent with a suggestive paternal influence on embryo development (Shoukir et al., 1998). The causes of the lower performance of ICSI in these conditions are unknown, but there is growing suspicion that an apoptosis-related cell death mechanism is a common denominator and an immediate cause of compromised 2640
sperm reproductive capacity. Indeed, increased percentages of spermatozoa with DNA strand breaks were found in semen samples with poor sperm count, motility and morphology (Sun et al., 1997; Barroso et al., 2000; Gandini et al., 2000; Muratori et al., 2000). Moreover, DNA-damaged spermatozoa may still be extremely motile (Aitken et al., 1998) and capable of achieving normal rates of fertilization with ICSI (Twigg et al., 1998). Current information about the occurrence and clinical significance of apoptosis-related phenomena in human male germ cells is very limited (Lin et al., 1997; Sinha Hikim et al., 1998; Tesarik et al., 1998a; Jurisicova et al., 1999). Even less is known about the implication of these phenomena in failures of ICSI with testicular spermatozoa. It has been shown previously that round spermatids recovered from the testis of men with complete spermiogenesis failure massively fall prey to apoptosis (Tesarik et al., 1998a). It has thus been suggested that apoptosis-related DNA damage may be responsible for the low efficacy of round spermatid conception reported in the literature (Tesarik et al., 1998b). Interestingly, in-vitro culture of testicular tissue from this category of patients facilitates the selection of healthy spermatids (Tesarik et al., 1999) and appears to improve clinical outcomes (Tesarik et al., 2000a). © European Society of Human Reproduction and Embryology
Testicular sperm culture in germ cell apoptosis
However, apoptosis may also seriously compromise the developmental potential of elongated spermatids and spermatozoa in patients in whom spermiogenesis arrest is not complete, and in whom some spermatids achieve the elongation phase. Here, a series of cases is described in which an attempt was made to improve the quality of spermatozoa recovered from men with these characteristics by in-vitro culture. Materials and methods Patients This study involved 11 infertile couples whose fertility problem was entirely caused by the male factor, without any female pathology being detected. The female age ranged between 26 and 34 years. All male partners of these couples suffered from non-obstructive azoospermia, the aetiology of which was unknown. All had normal 46,XY karyotype, no signs of varicocele, and no reported history of relevant infection or toxin exposure. All of these couples had undergone at least two unsuccessful assisted reproduction attempts with testicular sperm recovery and ICSI. Most of these previous attempts had been performed at other centres, and only three of them—realized in three different couples—were performed at the authors’ centre. Medical reports of the previous attempts mentioned the presence of very few spermatozoa in the testicular biopsy samples. Motile spermatozoa were found and used for ICSI in all of those attempts. In spite of relatively favourable fertilization rates (55–75%), most embryos available for transfer were of poor quality, and no pregnancy was established. Similar observations were made in the three previous attempts carried out at the authors’ centre. In addition, an analysis of germ cell apoptosis was performed using a modified deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method combined with immunocytochemical visualization of a germline marker (Tesarik et al., 1998a) on these occasions. This examination showed an unusually high percentage (⬎75%) of apoptotic cells in the round spermatid population. The representation of later post-meiotic stages was too low for a quantitative evaluation. Taken together, all cases involved in this study were characterized by non-obstructive azoospermia, at least two previous unsuccessful assisted reproduction attempts with testicular spermatozoa, the presence of spermatozoa in the testicular biopsy samples in all previous treatment attempts, and reportedly normal fertilization rates and poor embryo quality in the previous attempts. Because the selection of non-apoptotic cells is facilitated by germ cell in-vitro culture (Tesarik et al., 1999), a new treatment attempt was planned so as to perform testicular biopsy 2 days before oocyte recovery and to use cultureselected testicular spermatozoa for ICSI. Ovarian stimulation and oocyte recovery In 10 of the 11 cases involved in this study, ovarian stimulation was performed with recombinant FSH (Puregon; Organon, Madrid, Spain) after pituitary desensitization with triptorelin (Decapeptyl; Ipsen Pharma, Barcelona, Spain) started in the midluteal phase. In one case, a short protocol of pituitary desensitization was chosen, whereby the application of triptorelin began on the first day of the menstrual cycle followed by recombinant FSH treatment from day 2 onwards. The starting FSH dose (the first 4 days) was determined individually depending on age and basal serum FSH concentration, and was adapted from the day 5 of stimulation according to the dynamics of serum oestradiol concentration and the number and size of ovarian follicles determined by vaginal ultrasonography. The overall dose of FSH per stimulated cycle varied between 1800 IU and 3500 IU in individual patients. As soon as at least three follicles of ⬎18 mm
diameter were detected, ovulation was induced with 10 000 IU human chorionic gonadotrophin (HCG) (Profasi; Serono, Rome, Italy). Oocytes were recovered by transvaginal ultrasound-guided follicle aspiration performed 36 h after HCG injection. Testicular tissue recovery and in-vitro culture Samples of testicular tissue were obtained by open testicular biopsy from multiple sites in both testes. Tissue samples were disintegrated mechanically by stretching between two sterile microscope slides. After original inspection, individual samples obtained from the same patient were mixed together and homogenized by repeated aspirations into a tuberculin syringe. A small portion (~15–20% of total tissue volume) was further treated with collagenase I and elastase (Sigma Chemical Co., St Louis, Missouri, USA) as described (Tesarik et al., 1998c) to facilitate disintegration into single cells of the seminiferous tubule segments remaining intact after the mechanical treatment of the tissue. The enzymatically digested testicular tissue was then washed free of enzymes and processed for the evaluation of apoptosisrelated cell damage (see below). Most of each original sample (80–85% of tissue volume) was allocated to in-vitro culture. This tissue was not exposed to any enzymatic treatment at this step. Instead, it was placed into Gamete-100 medium (Scandinavian IVF Science, Gothenburg, Sweden) supplemented with 50 IU/l recombinant FSH (Puregon) and 1 µmol/l testosterone (water-soluble; Sigma, Cat. no. T-5035) and cultured at 30°C for 2 days (Tesarik et al., 1998d). At the end of culture, most of the tissue was used to recover spermatozoa for ICSI (see below), whereas a smaller part was treated with collagenase I and elastase (see above) and processed for the evaluation of apoptosisrelated cell damage. Evaluation of apoptosis-related cell damage These examinations involved the evaluation of DNA strand breakage and the detection of plasma membrane phosphatidylserine externalization. The former was performed with samples from five patients (patients 1–5) and the latter with samples from six patients (patients 6–11). Accordingly, each of the 11 patients involved in this study had one apoptosis-detection test performed with both freshly obtained and in-vitro-cultured samples. The paucity of spermatozoa in the testicular biopsy samples, together with the priority given to the recovery of sufficient numbers of spermatozoa for assisted reproduction, precluded the use of both examinations in each patient. DNA strand breakage Smears of disintegrated testicular tissue were processed by TUNEL using a commercially available kit (Apoptosis Detection System, Fluorescein) basically as recommended by the manufacturer (Promega, Paris, France), including the use of live–dead staining with propidium iodide to distinguish apoptosis from necrosis. The only difference from the recommended protocol was the use of glutaraldehyde instead of paraformaldehyde for smear fixation. This modification was based on previously reported observations showing that glutaraldehyde fixation increases the specificity of the TUNEL reaction for apoptosisrelated internucleosomal DNA strand breakage as opposed to nonspecific DNA fragmentation resulting from germ cell necrosis (Sinha Hikim et al., 1997; Tesarik et al., 1998a). This assay was used to evaluate the percentage of apoptotic cells in the overall population of living (excluding the supravital dye included in the TUNEL kit) testicular spermatozoa before and after in-vitro culture. Motile spermatozoa could not be distinguished with this technique because all observations were carried out in fixed smear preparations. Plasma membrane phosphatidylserine externalization Annexin-V-FLUOS Staining Kit (Boehringer Mannheim, Mannheim, Germany), based on the affinity of phosphatidylserine having migrated
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from the inner to the outer surface of the plasma membrane of apoptotic cells for fluorescein isothiocyanate (FITC)-labelled annexin V, was used to detect cytoplasmic changes typical of apoptosis in living testicular spermatozoa. Native preparations of enzymatically digested (see above) testicular tissue were incubated with the annexinV reaction mixture as recommended by the manufacturer. This test was used to evaluate the percentage of apoptotic cells in the motile subpopulation of living testicular spermatozoa before and after invitro culture. The distinction of motile spermatozoa was possible with this technique because the fluorescent marker was applied to living spermatozoa which continued to move during the microscopic examination. ICSI, embryo culture and transfer After in-vitro culture, further mechanical disintegration of the testicular tissue was carried out by crushing the remaining seminiferous tubule segments with sterile microscope slides, followed by repeated vigorous aspiration into a tuberculin syringe. Gamete-100 medium containing disintegrated testicular tissue was then placed in large (~50 µl) drops on plastic cell culture dishes and covered with light mineral oil (Ovoil; Scandinavian IVF Science). Motile spermatozoa swam out from the centre of the drops and accumulated at the boundary between the drop and the surrounding mineral oil where they were picked up with the use of a blunt-tip micropipette of internal diameter ~10 µm (Assisted Hatching Micropipets, Humagen, Charlottesville, Virginia, USA) and transferred to a small (~2 µl) drop of Gamete medium on another plastic dish which also carried, under mineral oil coverage, a drop of 10% polyvinylpyrrolidone in Gamete-100 medium (Scandinavian IVF Science) as well as several drops of Gamete-100 medium prepared to accommodate oocytes during the subsequent ICSI procedure. The selection of spermatozoa was carried out at laboratory temperature, while oocytes to be injected were maintained at 37°C in a CO2-supplied incubator. When a sufficient number of spermatozoa had been isolated, the dish with the selected spermatozoa was held in the CO2 incubator for a period of 15–30 min before placing the oocytes. Oocytes were incubated with 40 IU/ml hyaluronidase (Hyase, Scandinavian IVF Science) at 37°C for about 30 s to release the cumulus oophorus, followed by mechanical removal of the corona radiate by repeated aspiration in and out of a finely drawn glass Pasteur pipette. Metaphase II oocytes were then taken apart to be used for ICSI. Cumulus- and corona-free metaphase II oocytes were placed in drops of Gamete medium on the same culture dish, which also carried a drop of 10% polyvinylpyrrolidone and a drop of Gamete medium with previously selected motile spermatozoa (see above). ICSI was performed at 37°C using previously described techniques and instrumentation (Tesarik and Sousa, 1995). After ICSI, oocytes were cultured at 37°C in IVF medium (Scandinavian IVF Science) equilibrated with 5% CO2 in air for an additional 16–20 h, after which they were checked for signs of fertilization. Those oocytes that showed signs of normal fertilization (two pronuclei and two polar bodies) were placed into fresh IVF medium and returned to the CO2 incubator for an additional 26–30 h. Two to four cleaving embryos (2- to 6-cell stage) were transferred to the uterus of each patient. If supernumerary embryos were available, they were either frozen on the day of embryo transfer or cultured further in an attempt to obtain blastocysts to be frozen on the 5th or 6th day after ICSI. Statistical analysis Statistical significance of differences between percentages of spermatozoa showing apoptosis-related cell damage in before-culture and in
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after-culture samples was evaluated by a χ2-test using StatView II statistical package (Abacus Concepts, Berkeley, California, USA).
Results Assisted reproduction outcomes with in-vitro-cultured testicular spermatozoa The fertilization rate with in-vitro-cultured spermatozoa was acceptable in all 11 treatment attempts reported in this study (Table I). The overall mean fertilization rate was 71.6%. Out of 78 normally fertilized oocytes, all but three underwent at least one cleavage division by 2 days of in-vitro culture (Table I). Most of the cleaving embryos (49/75; 65.3%) reached the 4-cell stage by that time. Of the remaining 26 embryos, 14 were at the 2-cell stage, 10 at the 3-cell stage, and two at the 6-cell stage. Forty-two embryos (36 fresh, six frozen) were transferred to the uterus of the patients in the 11 infertile couples (Table I). The morphology grade of all transferred embryos was 3 or 4 according to a published classification (Bolton et al., 1989), where grade 4 denotes the best embryos and grade 1 the worst. Twenty-nine out of 39 supernumerary embryos were grade 2 or better, and were frozen on the day of embryo transfer. Only in two patients (patients 4 and 8) were there insufficient spare embryos with morphology that would justify immediate cryopreservation. Four supernumerary embryos from these patients were thus cultured further in an attempt to achieve the blastocyst stage. One of these embryos (patient 11) reached the blastocyst stage on day 5 after ICSI, and was cryopreserved. The remaining three embryos arrested during culture and were discarded. After fresh embryo transfer, performed in all 11 cases involved in this study, clinical pregnancy (detection of embryonic heartbeat) was established in six cases. Five pregnancies were singleton and one was twin (Table I). An additional biochemical pregnancy was lost before the detection of the embryonal sac. Cryopreserved embryos were transferred in three of the five cases in which an ongoing pregnancy did not occur after fresh embryo transfer (patients 1, 2 and 5). An additional singleton pregnancy was established in one of these patients (Table I). Incidence of apoptosis-related cell damage in total-living and motile sperm populations from before-culture and afterculture samples The occurrence of apoptosis-related DNA strand breakage was analysed in before-culture and after-culture samples from patients 1–5. Very low numbers of living (supravital dyeexcluding) spermatozoa in these samples was a major limiting factor. Nonetheless, working with total numbers of 50–100 living spermatozoa per sample, the after-culture samples showed a significantly lower percentage of spermatozoa with damaged DNA as compared with the before-culture samples in all five cases (Table II). The occurrence of apoptosis-related translocation of phosphatidylserine from the inside to the outside of the plasma membrane was assessed in before-culture and after-culture samples from patients 6–11. In spite of the low numbers of
Testicular sperm culture in germ cell apoptosis
Table I. Fertilization, embryo cleavage and pregnancy outcomes with in-vitro-cultured testicular spermatozoa in 11 azoospermic patients with severe testicular sperm apoptosis and two or more previous ICSI failures Patient no.
1 2 3 4 5 6 7 8 9 10 11
Oocytes
Cleaved embryos
Pregnancy
Injected
Fertilizeda
Total formed
Transferred/ frozen
Fresh embryos
Frozen embryos
10 12 11 6 8 10 8 9 15 13 7
7 7 9 5 6 7 6 5 11 9 6
7 7 8 5 6 6 6 5 10 9 6
3/3 3/4 4/2 4/0 3/3 3/3 3/3 3/0 3/6 4/5 3/1
No No Singletonb Twinb Biochemical Singletonb Singletonb No No Singletonb Singletonb
Singletonb No Not transferred – No Not transferred Not transferred – Not transferred Not transferred Not transferred
aOnly normal fertilization bNormal babies born.
(two pronuclei and two polar bodies) is taken into account.
Table II. Frequency of apoptosis-related DNA damage among living spermatozoa in before-culture and after-culture samples as determined by the TUNEL assay Patienta
1 2 3 4 5 Total
% spermatozoa with DNA damage Before-culture
After-culture
99/100 (99) 89/94 (95) 94/100 (94) 93/97 (96) 95/100 (95) 470/491 (96)
27/85 (32) 8/52 (15) 45/100 (45) 13/52 (25) 17/81 (21) 110/370 (30)
P
⬍ ⬍ ⬍ ⬍ ⬍ ⬍
0.001 0.001 0.01 0.001 0.001 0.001
Values in parentheses are percentages. aPatient identification numbers correspond to those in Table I.
Table III. Frequency of apoptosis-related plasma membrane phosphatidylserine externalization among motile spermatozoa in beforeculture and after-culture samples as determined by the annexin-V binding assay Patienta
6 7 8 9 10 11 Total
% spermatozoa with phosphatidylserine externalization Before-culture
After-culture
P
15/19 (79) 21/24 (88) 11/12 (92) 4/4 (100) 8/10 (80) 6/9 (67) 65/78 (83)
1/11 (9) 0/7 (0) 1/33 (3) 1/15 (7) 2/24 (8) 1/19 (5) 6/109 (6)
⬍ ⬍ ⬍ ⬍ ⬍ ⬍ ⬍
0.05 0.05 0.001 0.05 0.01 0.05 0.001
Values in parentheses are percentages. aPatient identification numbers correspond to those in Table I.
motile spermatozoa available for this analysis, a significant reduction of the percentage of spermatozoa with plasma membrane phosphatidylserine externalization was evident in each case (Table III).
Discussion The origin and mechanism of apoptosis-related cell damage, such as DNA strand breakage or plasma membrane phosphatidylserine externalization, in mammalian spermatozoa is poorly understood (Sakkas et al., 1999a). Many of the proteins known to mediate apoptosis in somatic cells, including proteins of the Bcl-2 family (Kojima et al., 2001), caspases (BlancoRodriguez and Martinez-Garcia, 1999) or Fas (Sakkas et al., 1999b) have been detected in male germ cells. Some of these proteins play a physiological role in the process of spermatogenesis, as demonstrated by the development of spermatogenesis disorders and infertility in Bax-knockout mice (Knudson et al., 1995) and by the physiologically restricted expression of caspase-1, c-jun, p53 and p21 in the cytoplasmic caudal tags of early elongating spermatids that subsequently detach during spermiogenesis by means of a subcellularly localized apoptotic process leaving out the spermatid nucleus (Blanco-Rodriguez and Martinez-Garcia, 1999). On the other hand, apoptosis is also involved in the removal of damaged germ cells or those germ cells in which further differentiation has been pathologically arrested, as evidenced by increased proportions of apoptotic germ cells in testicular biopsy samples from patients with complete spermiogenesis failure (Tesarik et al., 1998a) and in round spermatids of mice with targeted disruption of the Pp1cγ gene representing an animal model for human primary testicular failure (Jurisicova et al., 1999). The mechanism of apoptotic DNA damage in immature germ cells in the diseased human testis is poorly understood. Even less is known about the mechanism of apoptosis in mature spermatozoa. The incidence of DNA damage among ejaculated spermatozoa has been shown to be inversely correlated with several parameters reflecting semen quality, such as sperm count (Sun et al., 1997; Oosterhuis et al., 2000), motility (Sun et al., 1997; Glander and Schaller, 1999; Muratori et al., 2000; Oosterhuis et al., 2000) and morphology (Sun et al., 1997). Moreover, high frequencies of apoptotic spermatozoa in the ejaculate have been shown to be associated with increased sensitivity of spermatozoa to treatments inducing 2643
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DNA denaturation (Gorczyca et al., 1993; Aravindan et al., 1997) and with decreased fertilization (Sun et al., 1997; Host et al., 2000a), although other data have shown clearly that spermatozoa exhibiting significant DNA damage may still be capable of fertilizing the oocyte (Aitken et al., 1998; Host et al., 2000b). Hence, DNA damage appears to represent the end result of various testicular pathologies (Gandini et al., 2000). However, apoptosis-related proteins are shed from developing spermatids during residual body extrusion (BlancoRodriguez and Martinez-Garcia, 1999), so that morphologically normal mature spermatozoa are likely to be deficient in molecules mediating the classical apoptotic pathway. Moreover, the compact structure of sperm chromatin compromises its susceptibility to digestion by apoptosis-activated nucleases. Also, DNA fragmentation in human spermatozoa is not correlated with submicroscopic characteristics typical of somatic cell apoptosis (Muratori et al., 2000). These observations support the idea that apoptosis of mature spermatozoa proceeds through an atypical pathway (Sakkas et al., 1999b) which may not involve the caspase-dependent suicide mechanism and which appears to bear some similarity to apoptosis in chicken erythrocytes (Weil et al., 1998). In view of these considerations, the high incidence of apoptosis among testicular spermatozoa in cases reported in this study may be best explained by a disturbance of Sertoli cell–germ cell interaction. Previous studies have shown high percentages of apoptotic primary spermatocytes and round spermatids in men with severe testiculopathies (Tesarik et al., 1999, 2000b). Apoptotic round spermatids, however, are unlikely to complete spermiogenesis and develop to spermatozoa. Apoptotic testicular spermatozoa can thus be expected to have developed from germ cells that still had not been apoptotic at the round spermatid stage. The physiological, topographically restricted activation of the apoptotic process during spermiogenesis (Blanco-Rodriguez and Martinez-Garcia, 1999) may turn into a pathological condition if the cytoplasmic area concerned is not engulfed by adjacent Sertoli cells in due time. Instead of forming a residual body, this cytoplasmic area may remain associated with the spermatids, and the locally activated apoptotic machinery may extend its influence to other cell regions, including the nucleus. Partial mechanical disintegration of the seminiferous tubules, followed by in-vitro culture, appears to facilitate the separation of germ cells from Sertoli cells. Moreover, post-meiotic germ cell differentiation has been shown to be accelerated during in-vitro culture in media containing high concentrations of FSH and testosterone (Tesarik et al., 1998d, 2000c). This situation may promote the detachment of the pathologically retained residual body from the elongating spermatids and thus overturn the pathological extension of the physiologically restricted apoptotic process. Moreover, the enrichment of the post-culture living sperm population in non-apoptotic spermatozoa is likely to be at least partly attributable to death and disintegration of some spermatozoa that were apoptotic before culture. Hence, selective pressure against sperm apoptosis during 48 h of in-vitro culture may be independent of in-vitro acceleration of spermiogenesis which has been described to occur, under the same culture conditions, in a 2644
limited subpopulation of post-meiotic germ cells (Tesarik et al., 1998c,d). In practical terms, this pilot study confirms the conclusions of a previously published case report (Tesarik et al., 2000b), and shows that massive apoptosis of post-meiotic germ cells may become a new indication for testicular tissue in-vitro culture, even when mature spermatozoa can already be retrieved from the fresh testicular biopsy samples.
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