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2015, Volume 31, Number 2, Page(s) 111-118
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DOI: 10.5146/tjpath.2015.01303
Chromosome Abnormalities Identified in 457 Spontaneous Abortions and Their Histopathological Findings
Sezin YAKUT1, Havva Serap TORU2, Zafer ÇETİN3, Deniz ÖZEL4, Mehmet ŞİMŞEK5, İnanç MENDİLCİOĞLU5, Güven LÜLECİ1
1Department of Medical Biology and Genetics, Akdeniz University, School of Medicine, ANTALYA, TURKEY
2Department of Pathology, Akdeniz University, School of Medicine, ANTALYA, TURKEY
3Department of Medical Biology and Genetics, SANKO University, School of Medicine, GAZİANTEP, TURKEY
4Department of Biostatistics and Medical Informatics, Akdeniz University, School of Medicine, ANTALYA, TURKEY
5Department of Obstetrics and Gynecology, Akdeniz University, School of Medicine, ANTALYA, TURKEY
Keywords: Chromosomal abnormalities, Cytogenetic abnormalities, Pathology, Spontaneous abortion
Abstract
Objective: About 15% of clinically recognized pregnancies result in spontaneous abortion in the first trimester and the vast majority of these are the result of chromosome abnormalities. Studies of chromosomal constitutions of first trimester spontaneous abortions have revealed that at least 50% of the abortions have an abnormal karyotype. In this study we aimed to report the single centre experience of anomalies detected in spontaneous abortions.

Material and Method: We present rare numerical and structural cytogenetic abnormalities detected in spontaneous abortion materials and the histopathological findings of rest material of abortion specimens in our study population.

Results: Among 457 cases, 382 were successfully karyotyped while cell culture of 75 cases failed. Cytogenetic abnormalities were detected in 127 of 382 cases (33.24%). Autosomal trisomies were the predominant chromosomal abnormalities with a frequency of 48.8%. Structural chromosomal abnormalities were infrequent in conception materials. The mean age of the mothers was highest in trisomy group, the difference being significantly important (ANOVA p< 0.001). The most frequent chromosomal abnormalities were Turner syndrome, triploidy and trisomy of chromosome 16 followed by trisomy of chromosomes 22 and 21 and tetraploidy. Double trisomies and structural chromosomal abnormalities were rare. Trisomies were more frequent in advanced maternal age.

Conclusion: Detection of chromosomal abnormalities in spontaneous abortion materials is very important to clarify the causes of loss of pregnancy. Detection of structural chromosomal abnormalities in the cases and their carrier parents can provide proper genetic counseling to these families. These families can be directed towards pre-implantation genetic diagnosis to prevent further pregnancies with complications.

Introduction
About 15% of clinically recognized pregnancies result in spontaneous abortion (SAB) in the first trimester and the vast majority of these are the result of chromosome abnormalities1-3. Studies of chromosomal constitutions of first trimester spontaneous abortions have revealed that at least 50% of abortions have an abnormal karyotype4-6. There are several etiologies that might be associated with pregnancy losses including endocrine, immunological, environmental factors, infections, anatomic malformations and genetic abnormalities7. The most common chromosomal abnormality observed within first trimester spontaneous abortions is single trisomies8. Most clinically recognizable SABs occur between 7 and 11 weeks of gestation. Around 50% of spontaneous abortions are caused by de novo aneuploidy or polyploidy due to meiotic or post zygotic mitotic error, de novo unbalanced rearrangements, and unbalanced segregation products of the parental balanced translocations. Unbalanced chromosome constitution could affect placental development resulting in pregnancy failure9,10. Carriers of balanced reciprocal translocations have a high reproductive risk of conceiving chromosomally abnormal embryos as a result of imbalances during meiosis, leading to recurrent abortions or birth of affected children3,11. Chromosomal abnormalities can be detected by using conventional cytogenetic analysis. Evaluation of chromosomal abnormalities in pregnancy losses is important to understand the associations between chromosomal abnormalities and pregnancy losses and to provide proper genetic counseling to the parents. Histopathological evaluation of abortion material is also important because it may not be possible or reasonable to undertake complex cytogenetic studies of spontaneous abortion on a routine basis because of the expense and as it adds little to management.

We present here rare numerical and structural cytogenetic abnormalities detected in spontaneous abortion materials and the histopathological findings of rest material of abortion specimens in our study population.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Methods
    In our eleven-year experience, we had a total number of 457 miscarriage cases. Conception products were provided to Department and Medical Biology and Genetics by the department of the Obstetrics and Gynecology of the Akdeniz University Hospital to perform conventional cytogenetic analysis and the rest material of miscarriage samples were sent to pathology department for histopathological evaluation. Gestational ages varied from 7 to 36 weeks.

    Detailed reproductive histories were obtained from the families including gestational age, maternal age and outcome of previous pregnancies. To avoid maternal blood contamination, miscarriage samples containing chorionic villi were washed three times in physiological serum saline solution. Fetal or fetus-derived extra-embryonic tissues were identified and dissected from surrounding maternal deciduas. Selected tissue samples were minced and cultivated in T-25 tissue culture flasks including 5 ml Amniopan and Amniogrow complete mediums (Biotech, Cytogen), 0.05 ml penicillin-streptomycin solution (Biological Industries) and 0.05 ml L-Glutamine (Biological Industries). Metaphase chromosomes were harvested and G banded by GTG banding following standard procedures. In each case, at least twenty metaphase plates were evaluated by light microscopy.

    The rest miscarriage material of 76 of 457 cases was sent to the pathology department. Formalin-fixed and paraffinembedded blocks were sectioned in 3μ thickness and stained with hematoxylin eosin. These hematoxylin eosinstained sections were examined under the light microscope by an experienced pathologist.

    Descriptive analyses were given as frequency, percentage, mean and Standard Deviation (SD). The Pearson Chisquare test was used for analysis of categorical data. For comparing the age difference between the chromosomal abnormality groups, the ANOVA (Analysis of Variance) test was used and after finding a significant difference the Bonferroni test was used for pair-wise comparison. ROC (Receiver Operating Characteristic) analysis was used for differentiating the chromosomally abnormal group from the normal group according to age. For statistical analyses, the SPSS 18.0 package software was used. p < 0.05 was accepted as statistically significant.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Results
    Among the 457 cases, 382 were successfully karyotyped (culture success rate: 83.58%) while the cell culture of 75 cases failed (culture failure rate: 16.42%). Cytogenetic abnormalities were detected in 127 of 382 cases (33.24%). We included 257 cases of 382 karyotyped cases in our study group. This study group consisted of 127 cases of the karyotypically abnormal group and 130 cases of the control group with normal karyotype (with ages and abortion rates similar to the karyotypically abnormal group). The design of our study is given in Table I. The mMean maternal age in the karyotypically abnormal cases was 31.11 years (Standard Deviation (SD) ± 5.49) and the mean gestational age was 9.44 weeks (SD ± 3.39).


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    Table I: Scheme of study design

    Normal chromosomal constitution was detected in 255 cases (66.76%). The female/male sex ratio of the cases with a normal karyotype was 1.74 (162 females/93 males). The frequency of chromosomal abnormalities was higher in the group composed of cases with an age over 35 years than in younger cases (41.86% vs 30.67%). Chromosome abnormalities were more frequent in first trimester conceptions than second trimester conceptions (43.6% vs 7.77%). Turner syndrome and trisomy 16 were the most frequent abnormalities in the cases aged below 35 years of age, whereas trisomy 16 and trisomy 21 were the most frequent abnormalities in the cases aged over 35 years.

    Autosomal trisomies were the predominant chromosomal abnormalities with a frequency of 48.8% of all chromosome abnormalities, followed by 45, X, (n: 21, 16.5%), triploidies including mosaics (n: 17, 13.38%), tetraploidies (n: 7 cases; 5.5%), double or triple trisomies of various chromosomes (n: 6 cases, 4.72%), and XY/XX/XXY mosaicism (n: 1 case, 0.78%). The most common trisomies were trisomy 16 (n: 16, 12.7%), 22 (n: 10, 7.8%), 21 (n: 7, 5.5%), 13 (n:4, 3.1%) and 10 (n:4, 3.1 %) (Table II). Among the triploid cases, 12 cases had 69, XXY (one of them had an associated anomaly) and 5 cases were 69, XXX karyotypes (3 of them had an associated anomaly). 4 cases with tetraploidy had the 92, XXYY karyotype whereas 3 cases had the 92, XXXX karyotype.


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    Table II: Number and percentage of chromosomally abnormal group

    Karyotype descriptions of the cases with double trisomies were 48,XY,+7,+21, 48,XX,+16,+21, 48,XX,+13,+15, 48,XY,+2,+21. Trisomies of chromosomes 8, 16 and 21 were observed in one case. Also, trisomies of chromosomes 8, 12, 18, 20 were observed in another case.

    Structural chromosomal abnormalities were infrequent in conception materials, and some rare structural abnormalities including de novo structural chromosome abnormalities such as derdic13(13;18)(p11.1;p11.1) leading to partial trisomy 18p11.1-pter, del(18)(p11.2-pter) and del(7)(q22q32) were found. Unbalanced products of the parental balanced Robertsonian translocations were observed in four cases. In case 10, a derivative chromosome from adjacent-1 segregation of the paternal balanced reciprocal translocation t (6; 13) (p23; q12) resulted in partial monosomy 6p23-pter and partial trisomy 13q12- qter regions. In case 11, de novo der5 t(5;13) (p15; q12) resulted in partial monosomy 5p15-pter and partial trisomy 13q12-qter regions. Case 12 had interchange trisomy 7, resulting from 3:1 segregation of the familial reciprocal translocation t(5;7) (q13:p11.2). Co-existence of trisomy 16 and a familial transmitted balanced translocation t(1;5) (p22;q13) was observed in case 13 (Table III).


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    Table III: Rare numerical and structural chromosomal abnormalities detected in spontaneous abortion materials

    In cytogenetically abnormal groups; the mean age of the mothers was 28.57 (SD: ±4.79) years in the Turner group, 32.95 (SD: ± 5.01) in the trisomy group, 29, 29 (SD: ± 5.19) in the triploid and tetraploid group, and 28.42 (SD: ± 6.40) in the structural abnormalities group. The mean age of the mothers was highest in the trisomy group and the difference was significant (ANOVA p< 0.001). In the chromosomally abnormal group 8 of 21 Turner cases (38.1%), 37 of 70 trisomy cases (52.9%), 10 of triploidy-tetraploidy cases (41.7%), and 8 of 12 structural anomaly cases (66.7%) had a previous spontaneous abortion history. Statistically there was no significant difference between chromosomally abnormal cases (Chi-square, p=0.332) (Table IV). The mean age of the chromosomally normal 130 cases was 30.57 (SD: ± 5.31) years. The cut-off age for trisomies was 30 years (criterion values and coordinates of ROC curve are given in Figure 1).


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    Table IV: Mean age and abortus history of each karyotypically abnormal cases


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    Figure 1: ROC analysis of ages determining cut off age of trisomy ROC: Receiver Operating Characteristic, PPV: Positive Predictive Value, NPV: Negative Predictive Value, OR: Odds Ratio, CI: Confidence Interval

    Histopathological examination was performed for 72 cases and 53 cases (73.6%) had nonspecific changes (such as perivillous fibrin, hydropic villi), 11 cases (15.3%) were exaggerated placental site and 8 cases (11.1%) were incomplete mole hydatidiform. Exaggerated placental site cases cytogenetically consisted of 3 (27.3%) Turner syndrome, 1 (9.1%) trisomy 10, 1 (9.1%) trisomy 9, 2 (18.2%) tetraploidy and 2 (18.2%) rare trisomy cases. Partial mole hydatidiform cases cytogenetically consisted of 2 each of triploidy and structural anomaly, and one each of Turner syndrome, Trisomy 16, Trisomy 13 and double trisomy cases. There was no clinically significant relationship between histopathological diagnosis and cytogenetic anomaly.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Discussion
    The most frequent chromosomal abnormalities were Turner syndrome, triploidy and trisomy of chromosome 16 followed by trisomy of chromosomes 22 and 21 and tetraploidy in our study population while double trisomies and structural chromosomal abnormalities were rare. These findings were in concordance with the literature7,12. We detected several rare double trisomies and structural chromosomal abnormalities in spontaneous abortion materials.

    Double trisomy is a rare and could be detected in nearly 1.2% of the karyotyped spontaneous miscarriage materials with inter-institutional variability ranging between 0.80- 2.64 percent13,14. In our study, frequency of the double trisomy was 1.3% and in concordance with the literature. After the first report of a patient with Down-Klinefelter syndrome, more than 385 double trisomies have been reported14. Double trisomies were more frequent in cases with advanced maternal age greater than 35 years. The mean age of our cases with double or multiple trisomies was 35.6 years. Autosomal chromosomes frequently observed in single trisomies such as chromosomes 8, 13,15,16,18 and 21 and sex chromosomes were also the most frequently involved chromosomes in double trisomies in spontaneous abortions. According to the recent reviews, double trisomy of chromosomes X&22 was the first while trisomy 2&21 and trisomy 7&21 were the second reported cases in the literature. Triple or multiple trisomies are also rare with a frequency of 0.05% in spontaneous abortion materials15. This figure was 0.52% in our study population.

    Trisomies are more frequent in advanced maternal age16. The maternal age in the trisomy group was higher than the other chromosomally abnormal groups and the chromosomally normal group in our study as in the literature. The cut-off value for maternal age that increases the risk of trisomy was 30 years. The prenatal evaluation of a fetus where the maternal age is over 30 years should be more detailed because the estimated trisomy ratio is higher than the maternal age under 30 years. The most common trisomy is trisomy 21 in all pregnancy materials and live births17,18. It is known that the most common trisomy in spontaneous abortion is trisomy 1619. In our study, trisomy 16 was the most common abnormality in the trisomy group. Our cases were in the missed abortion group and this may show that trisomy 21 usually does not cause missed abortion as much as trisomy 16 and 22, but we can not say the same thing for the other trisomy groups because they are actually rare in all pregnancy materials13,18.

    Reciprocal translocations are the most common structural chromosomal rearrangements in humans with an estimated incidence of 1:712 in newborns20. In meiosis, the translocated chromosomes might be segregated in different segregation modes resulting in different chromosomal constitutions in gametes. Only alternate segregation results in normal or balanced gametes. Analysis of meiotic segregation modes by FISH in pre-implantation embryos from pre-implantation genetic diagnosis cycles showed that 2:2 segregation was the predominant segregation mode (59.1%) followed by, 3:1 segregation (22.0%), and 4:0 segregation (2.0%). In the 2:2 segregations, incidence of adjacent-1 (26.8%) was higher than that of alternate (22.4%) or adjacent-2 (6.1%) segregation11. Most of the embryos with unbalanced genetic content were eliminated by spontaneous abortions and still births21. In case 10, der6 t (6;13) (p23;q12) had developed from adjacent-1 segregation of the paternal translocation and resulted in partial trisomy 13q13-qter region and concomitant monosomy of the 6p23-pter. However, in case 11, the der5 t(5;13) (p15;q12) derivative chromosome was de novo and resulted in partial trisomy 13q12-qter and monosomy 5p15-pter, The rate of interchange trisomy resulting from 3:1 segregation was higher in balanced translocations with acrocentric chromosomes with a frequency of 9.5% than that without acrocentric chromosomes at 4.3%11. To our knowledge, our case 12 is the first case with interchange trisomy of the chromosome 7 detected in spontaneous abortion material resulting from 3:1 segregation of the familial translocation t(5;7)(q13;p11.2).

    The coexistence of a reciprocal translocation and chromosomal aneuploidy in an individual is also a rare event and several reciprocal translocation carrier Down syndrome patients have been reported22-24. In some of the cases, an inter-chromosome effect between these two events has been suggested25. To the best of our knowledge, this is the first report of the coexistence of trisomy 16 and familial transmitted balanced reciprocal translocation t(1;5) (p22; q13) detected in spontaneous abortion material.

    Another rare case had interstitial deletion of the q22-q32 band interval of the chromosome 7 and was spontaneously aborted at 9 weeks gestation. Intermediate interstitial deletion of chromosome 7 spanning from q22 to q31 bands is rare and causes multiple congenital malformations in the affected children26,27. Only a few rare prenatally detected cases presenting with fetal growth retardation and ultrasonographic findings such as cranial malformations, syndactyly in the lower extremities, renal pelvic dilatation, elevated nuchal fold thickness and cardiac malformations have been reported27-29.

    The evaluation of spontaneously aborted specimens has changed greatly and histopathological evaluation is not enough for a better understanding of the pathogenesis of the defects in aborted specimens. It is well known that it is necessary to know the chromosomal constitution19. In our study there was no significant relationship between histopathological diagnosis and chromosomal abnormality. The number of histopathologically evaluated cases are few and the literature and our findings suggest that histopathological evaluations provide limited data about the pathogenesis of the spontaneous abortions19.

    In conclusion, detection of chromosomal abnormalities in spontaneous abortion materials is very important to clarify the causes of pregnancy losses. Detection of structural chromosomal abnormalities in the cases and their carrier parents can provide proper genetic counseling to these families. These families can be directed towards pre-implantation genetic diagnosis to prevent further pregnancies with complications.

    FUNDING SOURCE
    This study was supported by the Akdeniz University Scientific Research Project Management Foundation, Antalya, Turkey.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
  • References

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    2) Baghbani F, Mirzaee S, Hassanzadeh-Nazarabadi M. Association of heteromorphism of chromosome 9 and recurrent abortion (ultrasound diagnosed blighted ovum): A case report. Iran J Reprod Med. 2014;12:357-60.

    3) R ajasekhar M, Gopinath PM, Sreelakshmi K, Satyamoorthy K. A cytogenetic study of couples with miscarriages: An experience from Manipal Referral Centre. Int J Hum Genet. 2013;13:93-7.

    4) Blumberg BD, Shulkin JD, Rotter JI, Mohandas T, Kaback MM. Minor chromosomal variants and major chromosomal anomalies in couples with recurrent abortion. Am J Hum Genet. 1982;34:948-60.

    5) Dewhurst J. Fertility in 47, XXX and 45, X patients. J Med Genet. 1978;15:132-5.

    6) Hassold T, Chen N, Funkhouser J, Jooss T, Manuel B, Matsuura J, Matsuyama A, Wilson C, Yamane JA, Jacobs PA. A cytogenetic study of 1000 spontaneous abortions. Ann Hum Genet. 1980;44:151-78.

    7) Warren JE, Silver RM. Genetics of pregnancy loss. Clin Obstet Gynecol. 2008;51:84-95.

    8) Hassold T, Jacobs P, Kline J, Stein Z, Warburton D. Effect of maternal age on autosomal trisomies. Ann Hum Genet. 1980;44:29-36.

    9) Qumsiyeh MB, Kim KR, Ahmed MN, Bradford W. Cytogenetics and mechanisms of spontaneous abortions: Increased apoptosis and decreased cell proliferation in chromosomally abnormal villi. Cytogenet Cell Genet. 2000;88:230-5.

    10) L junger E, Cnattingius S, Lundin C, Anneren G. Chromosomal anomalies in first-trimester miscarriages. Acta Obstet Gynecol Scand. 2005;84:1103-7.

    11) L im CK, Cho JW, Song IO, Kang IS, Yoon YD, Jun JH. Estimation of chromosomal imbalances in preimplantation embryos from preimplantation genetic diagnosis cycles of reciprocal translocations with or without acrocentric chromosomes. Fertil Steril. 2008;90:2144-51.

    12) Goddijn M, Leschot NJ. Genetic aspects of miscarriage. Baillieres Best Pract Res Clin Obstet Gynaecol. 2000;14:855-65.

    13) Huijsdens-van Amsterdam K, Barge-Schaapveld DQ, Mathijssen IB, Alders M, Pajkrt E, Knegt AC. Prenatal diagnosis of a trisomy 7/trisomy 13 mosaicism. Mol Cytogenet. 2012;5:8.

    14) Micale M, Insko J, Ebrahim SA, Adeyinka A, Runke C, Van Dyke DL. Double trisomy revisited-a multicenter experience. Prenat Diagn. 2010;30:173-6.

    15) R eddy KS. Triple aneuploidy in spontaneous abortions. Clin Genet. 1999;56:103-4.

    16) L amb NE, Yu K, Shaffer J, Feingold E, Sherman SL. Association between maternal age and meiotic recombination for trisomy 21. Am J Hum Genet. 2005;76:91-9.

    17) Allen EG, Freeman SB, Druschel C, Hobbs CA, O'Leary LA, Romitti PA, Royle MH, Torfs CP, Sherman SL. Maternal age and risk for trisomy 21 assessed by the origin of chromosome nondisjunction: A report from the Atlanta and National Down Syndrome Projects. Hum Genet. 2009;125:41-52.

    18) R obbins and Cotran Pathologic Basis of Disease. Kumar V, Abbas AB, Fausto N, Aster JC, editors. 8th ed. Philadelphia: Saunders Elsevier; 2010.

    19) Benirschke K, Burton GJ, Baergen RN. Pathology of the human Placenta. 6th ed. Berlin: Springer; 2012.

    20) N ielsen J, Wohlert M. Chromosome abnormalities found among 34,910 newborn children: Results from a 13-year incidence study in Arhus, Denmark. Hum Genet. 1991;87:81-3.

    21) L orda-Sanchez I, Diego-Alvarez D, Ayuso C, de Alba MR, Trujillo MJ, Ramos C. Trisomy 2 due to a 3:1 segregation in an abortion studied by QF-PCR and CGH. Prenat Diagn. 2005;25:934-8.

    22) Butomo IV, Mashkova MV. Double autosomal aberration: Trisomy 21 and familial reciprocal translocation t(10;12) (p14;q21). Tsitologiia. 1977;19:1291-6.

    23) Pazarbasi A, Demirhan O, Turgut M, Guzel I, Tastemir D. Inheritance of a translocation between chromosomes 12 and 16 in a family with recurrent miscarriages and a newborn with Down syndrome carrying the same translocation. Genet Couns. 2008;19:301-8.

    24) Garcia-Delgado C, Bahena-Martínez E, Aparicio-Onofre A, Guevara-Yañez R, Cervantes-Peredo A, Azotla-Vilchis OC, Estrada-Mena J, Luna-Angulo A, Villa-Morales J, Moran- Barroso VF. A familial reciprocal translocation t(1;15) in three generations identified in a regular trisomy 21 patient. Genet Couns. 2010;21:299-306.

    25) Oikawa K, Trent M, Lebovitz R. Familial balanced translocation 4p+/17q- as a suggested cause of primary trisomy-21 Down's syndrome. Arch Dis Child. 1977;52:890-3.

    26) Courtens W, Vermeulen S, Wuyts W, Messiaen L, Wauters J, Nuytinck L, Peeters N, Storm K, Speleman F, Nöthen MM. An interstitial deletion of chromosome 7 at band q21: A case report and review. Am J Med Genet A. 2005;134A:12-23.

    27) Cheong ML, Tsai MS, Cortes RA, Harrison MR. Intermediate interstitial deletion of chromosome 7q detected by first-trimester Down's syndrome screening. Fetal Diagn Ther. 2008;24:340-4.

    28) Y ilmaz Z, Eroglu D, Derbent M, Haberal AN, Lembet A, Sahin FI. Prenatal diagnosis of a partial monosomy 7q11-->q31 in a fetus with split foot. Fetal Diagn Ther. 2005;20:132-5.

    29) Hafen LB, Rose NC. Interstitial deletion of chromosome 7q and the lack of association with Down syndrome screening markers. Fetal Diagn Ther. 2010;28:47.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
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