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FISH y FRAGMENTACIN DE ADN: ASPECTOS PRCTICOS

R. Nez Calonge Clnica Tambre Madrid

FISH y fragmentacin ADN: nos sirven para algo en la prctica diaria?


En qu casos? Qu tcnica es mejor?

FISH DE ESPERMATOZOIDES
Es una tcnica molecular que nos permite detectar secuencias de ADN especficas en el ncleo del espermatozoide en estadio de interfase, mediante sondas de ADN fluorescentes, y determinar as su dotacin cromosmica.
sonda ADN secuencia cromosmica Descondensacin Desnaturalizacin Hibridacin

Visualizacin/interpretacin de seales

FISH: INTRERPRETACIN DE LAS SEALES


Haploide: presenta una seal nica para cada cromosoma analizado. Dismico: presenta dos seales para un cromosoma concreto. Diploide: presenta dos seales para cada cromosoma analizado. Nulismico: no presenta un cromosoma concreto o es un fallo de hibridacin, se considera que la incidencia de la nulisoma es igual a la de disoma.
Haploide 18 X

Disoma 18 YY Diploide 1818 YY

Diploide 1818 XY

Nulisoma X o Y

FISH DE ESPERMATOZOIDES
Determina la dotacin cromosmica, expresando el porcentaje de espermatozoides que presentan aneuploidas o disomas.
Se estima que el porcentaje de espermatozoides aneuploides en una poblacin control es del 6%, mientras que para los cromosomas X, Y, 13, 16, 18 y 21 es del 1,13 %

INDICACIONES DEL FISH

OATz severa Fallo de implantacin Aborto de repeticin Azoospermias secretoras Pacientes quimioterapia

CONTROVERSIAS DEL FISH


Uno de los problemas derivados del estudio del FISH radica en la interpretacin de resultados, ya que cada laboratorio posee sus controles especficos.

Pacientes con un porcentaje de aneuploidias mayor que los controles y que no han optado por el PGS han conseguido un embarazo normal.

QUE SABEMOS DEL FISH:


Segn la bibliografa, aumentan las aneuploidias:

Aborto de repeticin/ fallo de implantacin Azoospermia no obstructiva Post quimioterapia Relacionada con aumento de fragmentacin ADN ndice teratozospermia elevado

QUE SABEMOS DEL FISH:


Segn nuestra experiencia:

En ciclos con FISH alterado, no hay diferencias respecto a fecundacin, divisin y calidad embrionaria. Se obtienen gestaciones normales con FISH alterado, pero mayor nmero de abortos. No hay variabilidad intraindividuo

CONCLUSIONES FISH:

CUANDO
100% morfoanomalas o defecto predominante

FISH normal

FISH alterado

ICSI

DGP / Banco de semen

Fallo implantacin Aborto repeticin

DGP / Banco de semen

CONCLUSIONES FISH:

La verdadera utilidad del estudio de FISH en espermatozoide radica en los casos en los que la frecuencia de alteraciones cromosmicas es tan alta que la nica opcin vlida es emplear semen de donante.

FRAGMENTACIN DE ADN

FRAGMENTACIN DE ADN

Leucospermia

Aneuploidias

Estrs oxidativo

Fragmentacin cadena doble y cadena sencilla

Enciso et al, RBM, 2009.

En donantes frtiles se ha observado un aumento del ndice de fragmentacin entre las 4-6 horas de incubacin a 37C, con un aumento aproximado de 8% por hora.

Dinmica de la fragmentacin en muestras de semen antes y despus del swim-up agrupadas de acuerdo con el nivel de embarazo conseguido.

Existen indicios que indican que las dinmicas de fragmentacin de menor nivel basal de dao tras swim-up produciran embarazos con mayor facilidad

Fragmentacin de ADN

El nivel basal de SDF despus del swim-up es significativamente menor comparado con el semen antes de la recuperacin.. (T Student. p<0,001).

ESHRE 2009

Fragmentacin de ADN

La fragmentacin del ADN contina a lo largo del tiempo de forma similar antes y despus del swim-up.

Fragmentacin de ADN

Analizado desde un punto de vista dinmico el comportamiento de la fragmentacin conforme aumenta el tiempo transcurrido entre la descongelacin de la muestra y su determinacin, vemos un aumento progresivo de la fragmentacin conforme pasa el tiempo desde un 20,59% en el momento de la descongelacin a un 75,88% a las 72 horas

Fragmentacin de ADN

.8, CG els 1.0 05 61 72 17 vee /L of .5, ere

O-069 Effect of sperm DNA fragmentation on reproductive outcome with donated oocyte cycles R. Nunez - Calonge 1, J.A. Guijarro2, L. Ortega1, E. Olaya1, S. Cortes1, J. Gosalvez3, P. Caballero 1 1 Clinica Tambre, Reproduction Unit, Madrid, Spain Abstracts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 Jul y 6 July, 2011 2 Hospital Virgen de la Luz, Gynecology, Cuenca, Spain 3 Universidad Autonoma, Department of Biology, Madrid, Spain

ay are cy CG ed

ng ole ure a-

demographic characteristics or prestimulation parameters. The mean number of if this is a general trend or specic to certain patient populations or stimulation protocols. oocytes was D0: 9.3 6.3, D50: 8.5 4.4, D100: 8.6 shown 4.6 and D150: 11.3 5.7 fertility I ntroduction: Several studies have that male is affected by (p = 0.48) and the number of patients with embryo transfer was D0: 88%, D50: sperm DNA damage. When analyzing the molecular 93%, D100: 94% and D150: 77% (p = 0.48). The mean number of top-quality components of sperm, embryos per patient was D0: 0.8 1.2, D50: 0.5 0.7, D100: 1.2 1.7 and including DNA integrity and their relationship with embryo quality and IVF D150: 1.5 1.7 (p = 0.04). Positive s-hCG 14 days embryo replacement SELECTED ORAL COMMUNICATION SESSION success, female aetiology should beafter taken into account. Ovum donation is a was D0: 44%, D50: 33%, D100: 44% and D150: 39% (p = 0.92). All pregnanSESSION 19: ANDROLOGY useful with whichand to ongoing study the quality of 1012) spermwere used in assisted reproduc-- MALE AND SEMEN FACTORS cies were tool singleton gestations, pregnancies (week Monday 4 Julyassociated 2011 15:15 - 16:30 D0: 25%, D50: 27%, D100: 25% and 31% (p = 0.98) per cycle. Includtion treatments, because itD150: reduces the variability of oocyte quality ing thaw cycles, cumulated ongoing pregnancy rates were D0: 31%, D50: 33%, with female infertility. D100: 44% and D150: 39% (p = 0.89). On the day of hCG the mean numObjective: The of this study is to the effect on reproductive outber of intermediate (10 aim to 12 mm) follicles was D0: 4.4 quantify 3.9, D50: 4.5 4.8, O-069 from Effect of sperm DNA fragmentation on reproductive outcome D100: 3.3of 2.7 and D150: 2.5 Fragmentation 3.1 (p = 0.45). Steady state level s-hCG samples come Sperm DNA (SDF) in of sperm patients with donated oocyte cycles was reached on Day 6 of stimulation. On the day of hCG serum hCG levels undergoing intracytoplasmic sperm injection (ICSI) with frozen-thawed sperm, (IU/L) were D0: < 0.1, D50: 3.1 (2.63.6), D100: 5.5 (4.17.4) and D150: 11.0 1 R. in Nunez - Calonge , J.A. Guijarro2, L. Ortega1, E. Olaya1, S. Cortes1, using donated oocyte cycles from donors withwere proven fertility previous cycles. (8.913.6) (p < 0.001); the levels of s-androstendione (ng/mL) D0: 2.05 J. Gosalvez3, P. Caballero 1 (1.612.62), 3.21 (2.314.45), D100: (3.244.99) and D150: MaterialD50: and Methods: SDF4.02 was determined by4.61 analyzing aliquots of the Unit, Madrid, Spain 1 Clinica Tambre, Reproduction (3.506.07) (p < 0.01) and the s-progesterone (ng/mL) levels were D0: 0.72 2 same ejaculated sample that was used for ICSI. Frozen-thawed sperm Hospital Virgen samples de la Luz, Gynecology, Cuenca, Spain (0.580.89), D50: 1.03 (0.791.34), D100: 0.92 (0.791.08) and D150: 1.17 3 Universidad Autonoma, Department of Biology, Madrid, Spain employed in ICSI with donated oocytes (n = 70 samples) were analyzed before (0.821.67) (p = 0.03). S-estradiol will be presented later. The patients receivIntroduction: Several studies have shown that male fertility is affected by ing hCG supplementation were stratied by 33% and 66% percentiles into three taken and after swim-up. Semen aliquots (10 to 100 L) were from each sample sperm DNA damage. When analyzing the molecular components of sperm, groups according to the level of s-hCG on Day 6 of stimulation: 0.53.5 IU/L for processing by means of the SCD assay, using the Halosperm kit (Halotech including DNA integrity and their relationship with embryo quality and IVF (n = 16), 3.58.0 IU/L (n = 14) and 8.0 IU/L (n = 14). The mean number of DNA, SL, Madrid, Spain). success, female aetiology should be taken into account. Ovum donation is a top-quality embryos in the three groups was 0.5 0.9, 1.1 1.8 and 1.5 1.5, useful tool with which to study the quality of sperm used in assisted reproducA total of 70 ICSI cycles employing donated oocytes donors of proven respectively (p = 0.03). The ongoing pregnancy rates per started cycle were with tion treatments, because it reduces the variability of oocyte quality associated 31%, 21% and (p = 0.83).cycles were studied. Clinical pregnancy was conr med fertility in29% previous with female infertility. Conclusion: Supplementation with hCG from the rst day of stimulation may when the a gestational sac with fetal was detected by ultrasound 7study is to quantify the effect on reproductive outObjective: The aim ofat this increase number of top-quality embryos; even heartbeat doses up to 150 IU/day are come of Sperm DNA Fragmentation (SDF) in sperm samples from patients weeks of pregnancy. consistent with a high proportion of top-quality embryos and good pregnancy Spearman correlation for metric values undergoing intracytoplasmic sperm injection (ICSI) with frozen-thawed sperm, rates. Our In terms of top-quality embryos, doses from 100 to 150 IU/day of hCG primary outcome was the effect of DNA damage on clinical pregnancy using donated oocyte cycles from donors with proven fertility in previous cycles. may be optimal, but a proportion of patients with these doses have increased p:0073 p:0975 (CP). Welevels employed operating characteristic (ROC) curves to test the Material and Methods: SDF was determined by analyzing aliquots of the progesterone on the last receiver days of stimulation. T Student for of comparison 100% tax or less (graphics) same ejaculated samplethe that was used for ICSI. Frozen-thawed sperm samples predictive value SDF withbetween respect to achievement of pregnancy. We used employed in ICSI with donated oocytes (n = 70 samples) were analyzed before 70 cycles to determine thresholds of SDF. p:0044 p:0691 O-068 Does the addition of LH-activity to FSH make gonadotrophins and after swim-up. Semen aliquots (10 to 100 L) were taken from each sample Secondary outcomes fertilization rate (FR), cleavage rate (CR) and for processing by means of the SCD assay, using the Halosperm kit (Halotech more superior: a systematic review were and meta-analysis? DNA, SL, Madrid, Spain). embryo quality (EQ). Relationships between SDF and the FR, CR and EQ were A.M. Abou-Setta1, M.A. Aboulghar2, R.T. Mansour3, G.I. Serour 3, A total of 70 ICSI cycles employing donated oocytes with donors of proven compared H.G. Al-Inany2 using the Spearman rank correlation test and chi-square test. Correfertility in previous cycles were studied. Clinical pregnancy was conrmed 1

Sperm DNA fragmentation and fertilization rate, cleveage rate and embryo quality

Downloaded from http://humrep.oxfordjournals.org/ by Pedro Caballero on October 31,

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p:0860

Downloaded from http://humrep.oxfordjournals.org/ by Ped

% SDF
ANCOVA: FI mean for pregnancy: 2570% (IC: 2134-3002) FI mean for no pregnancy: 35,90% (IC: 3027-4153) difference of means: 10,193% (IC 95%: 2,97-17,41) p:0006 Marginal estimated means of F.I. for ANCOVA model: count: 569 mill/ml motility: 4388% fecondation tax: 8539% division tax: 9892% embryos 1-2: 7165%

cts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 Jul y 6 July, 2011

aphic characteristics or prestimulation parameters. The mean number of was D0: 9.3 6.3, D50: 8.5 4.4, D100: 8.6 4.6 and D150: 11.3 5.7 8) and the number of patients with embryo transfer was D0: 88%, D50: 100: 94% and D150: 77% (p = 0.48). The mean number of top-quality s per patient was D0: 0.8 1.2, D50: 0.5 0.7, D100: 1.2 1.7 and 1.5 1.7 (p = 0.04). Positive s-hCG 14 days after embryo replacement : 44%, D50: 33%, D100: 44% and D150: 39% (p = 0.92). All pregnanre singleton gestations, and ongoing pregnancies (week 1012) were yes %, D50: 27%, D100: 25% and D150: 31% (p = 0.98) per cycle. Includw cycles, cumulated ongoing pregnancy rates were D0: 31%, D50: 33%, 44% and D150: 39% (p = 0.89). On the day of hCG the mean numntermediate (10 to 12 mm) follicles was D0: 4.4 3.9, D50: 4.5 4.8, 3.3 2.7 and D150: 2.5 3.1 (p = 0.45). Steady state level of s-hCG ched on Day 6 of stimulation. On the day of hCG serum hCG levels were D0: < 0.1, D50: 3.1 (2.63.6), D100: 5.5 (4.17.4) and D150: 11.0 .6) (p < 0.001); the levels of s-androstendione (ng/mL) were D0: 2.05 .62), D50: 3.21 (2.314.45), D100: 4.02 (3.244.99) and D150: 4.61 .07) (p < 0.01) and the s-progesterone (ng/mL) levels were D0: 0.72 .89), D50: 1.03 (0.791.34), D100: 0.92 (0.791.08) and D150: 1.17

if this is a general trend or specic to certain patient populations or stimulation protocols.

SELECTED ORAL COM M UNI CATI ON SESSI ON SESSI ON 19: ANDROLOGY - M ALE AND SEM EN FACTORS M onday 4 July 2011 15:15 - 16:30

O-069 Effect of sperm DNA fragmentation on reproductive outcome with donated oocyte cycles R. Nunez - Calonge 1, J.A. Guijarro2, L. Ortega1, E. Olaya1, S. Cortes1, J. Gosalvez3, P. Caballero 1 1 Clinica Tambre, Reproduction Unit, Madrid, Spain Abstracts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 Jul y 6 July, 2011 2 Hospital Virgen de la Luz, Gynecology, Cuenca, Spain
3

Fragmentacin de ADN

Fresh F.I. p:0436 Capac F.I. p:0489 Count Movility Reference line

racts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 Jul y 6 July, 2011
if this is a general trend or specic to certain patient populations or stimulation Curva ROC protocols. rea bajo la curva:
IF fresco: 0682 (IC 95%: 0555-0809) IF capacitado: 0704 (IC 95%: 0580-0827)

graphic characteristics or prestimulation parameters. The mean number of es was D0: 9.3 6.3, D50: 8.5 4.4, D100: 8.6 4.6 and D150: 11.3 5.7 0.48) and the number of patients with embryo transfer was D0: 88%, D50: D100: 94% and D150: 77% (p = 0.48). The mean number of top-quality yos per patient was D0: 0.8 1.2, D50: 0.5 0.7, D100: 1.2 1.7 and : 1.5 1.7 (p = 0.04). Positive s-hCG 14 days after embryo replacement D0: 44%, D50: 33%, D100: 44% and D150: 39% (p = 0.92). All pregnanwere singleton gestations, and ongoing pregnancies (week 1012) were 5%, D50: 27%, D100: 25% and D150: 31% (p = 0.98) per cycle. Includaw cycles, cumulated ongoing pregnancy rates 1-Specificity were D0: 31%, D50: 33%, : 44% and D150: 39% (p = 0.89). On the day of hCG the mean numf intermediate (10 to 12 mm) follicles was D0: 4.4 3.9, D50: 4.5 4.8, : 3.3 2.7 and D150: 2.5 3.1 (p = 0.45). Steady state level of s-hCG eached on Day 6 of stimulation. On the day of hCG serum hCG levels ) were D0: < 0.1, D50: 3.1 (2.63.6), D100: 5.5 (4.17.4) and D150: 11.0 13.6) (p < 0.001); the levels of s-androstendione (ng/mL) were D0: 2.05 2.62), D50: 3.21 (2.314.45), D100: 4.02 (3.244.99) and D150: 4.61 6.07) (p < 0.01) and the s-progesterone (ng/mL) levels were D0: 0.72 0.89), D50: 1.03 (0.791.34), D100: 0.92 (0.791.08) and D150: 1.17 1.67) (p = 0.03). S-estradiol will be presented later. The patients receiv-

Sensitivity

SELECTED ORAL COM M UNI CATI ON SESSI ON SESSI ON 19: ANDROLOGY - M ALE AND SEM EN FACTORS M onday 4 July 2011 15:15 - 16:30

O-069 Effect of sperm DNA fragmentation on reproductive outcome with donated oocyte cycles R. Nunez - Calonge 1, J.A. Guijarro2, L. Ortega1, E. Olaya1, S. Cortes1, J. Gosalvez3, P. Caballero 1 1 Clinica Tambre, Reproduction Unit, Madrid, Spain Abstracts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 Jul y 6 July, 2011 2 Hospital Virgen de la Luz, Gynecology, Cuenca, Spain 3 Universidad Autonoma, Department of Biology, Madrid, Spain
demographic characteristics or prestimulation parameters. The mean number of

if this is a general trend or spe

Intra-individual variations in Sperm DNA Fragmentation in men from infertile couples compared with fertile sperm donors
R. Nez-Calonge,1 S.Corts,1 ,L.Ortega 1 E.Olaya, 1 A.Guijarro1 J.Goslvez, 2 P. Caballero1 1- CLINICA TAMBRE 2- Universidad Autonoma Madrid,

Resultados
Sperm DNA Fragmentation index
Mean DS 10.1 SD 9.8

PS

32.4

11.4

P<0.01 P=0.030

Results
SD intra-variability is only lower than the inter-individual in the group with <20% SDF.

SDF<20% Mean

SDF>20<30% SD interindividual

SDF>30% SD intraindividual

QUE SABEMOS DE LA FRAGMENTACIN DE ADN:


La fragmentacin de ADN aumenta: Aborto de repeticin/ fallo de implantacin Post quimioterapia Astenozoospermia Infecciones Estrs oxidativo Varicocele Edad paciente Tiempo de incubacin

QUE SABEMOS DE LA FRAGMENTACIN DE ADN:


La fragmentacin de ADN disminuye:

Abstinencia sexual Espermatozoides de testculo (?) Post swim-up- gradientes

QUE SABEMOS DE LA FRAGMENTACIN DE ADN:

No hay relacin con la tasa de fecundacin, divisin y calidad embrionaria Gran variabilidad intraindividuo en casos de IF elevado.

CONCLUSIONES FRAGMENTACIN ADN


Cundo determinar el ndice de fragmentacin?

Fallo de implantacin / aborto repeticin


Astenozoospermia/necrozoospermia Antes de donacin de ovocitos. Si el IF es elevado, repetirlo.

FISH y fragmentacin ADN: nos sirven para algo en la prctica diaria?


En qu casos?

Qu tcnica es mejor?

OATz severa Fallo implantacin/ aborto repeticin Pre-Qt Az secretora Asteno severa Donacin ovocitos

Fragmentacin Fragmentacin + FISH Fragmentacin + FISH FISH Fragmentacin Fragmentacin