Aula Indice de Fragmentação de DNA espermático: como avaliar e tratar. Conrado Alvarenga M.D. - XI Congresso Paulista de Medicina Reprodutiva, 2017 - mesa de andrologia.
2. Urologista formado pela Faculdade de Medicina da Universidade de São Paulo (USP),
especializado em Andrologia e Infertilidade Masculina. Membro do corpo clínico do Hospital
Sírio Libanês e da Divisão de Urologia do Hospital das Clínicas da Faculdade de Medicina da
USP
DR. CONRADO ALVARENGA
APOIO
4. 16%
Yearly decline in men’s
Sperm count in the West
SOURCE: HUMAN REPRODUCTION UPDATE
5. QUEDA DA FERTILIDADE
MASCULINA PELO MUNDO
The research = 26,600 men showed a "significant and continuous"
32.2% decrease in sperm concentration over 17 years
Sperm per millilitre of semen fell at about 2% a year between
1989 and 2005
In French men with an average age of 35, sperm count
reduced = 73.6 million/ml to 49.9 m/ml.
6. AUMENTO DO
IMC E INGESTA
DE GORDURA
SATURADA
AUMENTO DO
SEDENTARISMO
AUMENTO DOS
NÍVEIS DE
ESTRESSE
PIORA A
QUALIDADE DO
SONO
AUMENTO DOS
DISRUPTORES
ENDÓCRINOS
POLUENTES
AMBIENTAIS
HIPÓTESES PRINCIPAIS
QUEDA DA FERTILIDADE MASCULINA PELO MUNDO
7. QUAL A IMPORTÂNCIA DO ESTUDO DA
FRAGMENTAÇÃO DA CROMATINA ESPERMÁTICA ?
15% dos homens inférteis apresentam espermograma normal
8. Não é marcador de
fertilidade ou
infertilidade
Até 18% dos espg normais tem
elevados IFDNA
10. A integridade do DNA espermático = essencial para a correta
transmissão da informação genética
Alterações na cromatina ou dano direto ao DNA pode resultar
em infertilidade
Alterações na cromatina ou dano direto ao DNA
RESULTAM EM AUMENTO NO RISCO ABORTO
18-20% DOS ESPG NORMAIS TEM IFDNA ALTERADO
11. SURGEM PRIMEIROS TRABALHOS
Tempo mais longo para estabelecer uma gestação normal,
maior número de ciclos de Fertilização in vitro e aumento do
risco de abortamento espontâneo
FRAGMENTACAO DE DNA NO SEMEM
12.
13. Conclusions The SCSA analysis is a precise, timely and cost-effective
diagnostic tool to help manage a couple’s infertility treatment, whereas the
Comet and Tunel assays currently lack the consistency and reliability needed
in a clinical setting.
all idiopathic couples;
men older than 40 years, even if prior fertility;
men with known exposures to toxicants;
men with above mentioned pathologies; and
all couples presenting for infertility
Patients presenting with the following should have a DNA fragmentation test:
14. In the words of Dr. Larry Lipshultz, a well recognized urologist who is at the
forefront of modern urological practice at Baylor Medical College, ‘‘I perform
DNA fragmentation testing for all my patients who are undergoing IVF with ICSI
so that I can give them a general idea of how successful their cycles might be. If
the tests show greater than 26% DNA fragmentation, chances for poor embryo
development increase.’’
D.P. Evenson, R. Wixon / Theriogenology 65 (2006) 979–991
15.
16. Mean (SD), median (range) Control Subfertile men P
Mean no. of sperm analysed 7264 5212
DFI, % 8.4 (5.6), 5.0 (4–21) 22.8 (14.6), 17.0 (4–63) <0.001
Moderate DFI 6.4 (4.5), 4.0 (3–17) 16.4 (11.1), 13.0 (3–52) 0.001
High DFI 2.0 (1.3), 1.0 (1–4) 6.2 (6.1), 6.1 (0–26) 0.01
DNA FRAGMENTATION DATA FOR THE FERTILE CONTROL SUBJECTS AND SUBFERTILE MEN
17.
18. Halosperm may not be the definitive technique, if indeed one
exists, but its ease of use and interpretation facilitates its
potential incorporation into any basic research and clinical
laboratory in the world. This is in contrast to the more complex
SCSA. With greater access to a simple, reliable, and sensitive
sperm DNA fragmentation technique, perhaps conclusions about
the clinical validity of such assays will be made more swiftly.
19. 500
células
Normal Até 15% dos espermatozóides com DNA fragmentado
Moderado Entre 16 a 30% dos espermatozóides com DNA fragmentado
Elevada Acima de 30% dos espermatozóides com DNA fragmentado
Resultado – 12,8% (porcentagem de espermatozóides com DNA fragmentado)
Espermatozóides fragmentado / Espermatozóides analisados: 64 / 500
Metodologia – Dispersão da Cromatina Espermética (SCD Sperm Chromatin Dispertion) – HALOSPERM®
FRAGMENTAÇÃO DO DNA ESPERMÁTICO
20.
21. 16TRABALHOS 2.969 casais
Is there an association between high levels
of sperm DNA damage and miscarriage?
Significant increase in miscarriage in patients with
high DNA damage compared with those with
low DNA damage [risk ratio (RR) = 2.16 P < 0.00001]
22. Whether sperm deoxyribonucleic acid fragmentation has an effect on pregnancy and
miscarriage after in vitro fertilization/intracytoplasmic sperm injection: a systematic
review and meta-analysis.
OBJECTIVE:
To examine whether sperm DNA fragmentation has an effect on pregnancy and miscarriage after IVF
and/or intracytoplasmic sperm injection (ICSI).
DESIGN:
Systematic review and meta-analysis.
SETTING:
University-affiliated teaching hospital.
PATIENT(S):
Infertility patient(s).
INTERVENTION(S):
23. IncludedEligibillitySreeningIdentification Records identified through
database searching (n=401)
Studies included in quantitative
meta-analysis for miscarriage (=14)
Additional records identified
though other sources (n=0)
Records after duplicates
removed (n=400)
Records excluded (n=302)
Records screened (n=98)
Full-text articles excluded, with
reasons (=75)
Full-text articles assessed for
eligibility (n=23)
Studies included in quantitative
meta-analysis for pregnancy (n=16)
25. MENSAGENS
A grande maioria dos trabalhos publicados relacionando resultados da
reprodução assistida com índice de fragmentação de DNA
1
POSSUEM
NÚMERO
PEQUENO DE
PACIENTES
2
MÉTODOS
DIFERENTES DE
ANÁLISE E SELEÇÃO
NÃO HOMOGÊNEA
DOS CASOS
Fica claro e evidente que o dano ao DNA espermático afeta sim os resultados
da FIV/ICSI, principalmente nas taxas de gravidez clínica e de aborto
26. Ainda ha duvida e muitos
pontos a serem esclarecidos
Fertility and Sterility® Vol. 99, No. 1, January 2013 0015-0282
27. QUAIS SÃO OS TESTES DISPONÍVEIS
TUNEL assay 1993 Comet assay1996
Sperm chmatin sperm
structure (SCSA)
1980
Sperm chmatin
dispersion test (SCD)2003
Aniline blue, toluidine blue
1995
?
28. Métodos para avaliar a FDE
Direto
Indireto
Incorporação de probes no local da fragmentação
Ex: Tunel
Susceptibilidade das quebras à desnaturação em uma solução
ácida
Ex: Sperm chromatin structure assay (SCASA), sperm chromatin
dispersion test (SCD), Comet assay
29. Qual Teste Adotar em seu laboratório?
DIFICULDADE TÉCNICA E
TEMPO DE EXECUÇÃO
NECESSIDADE DE
EQUIPAMENTOS
COMPLEXOS E CAROS
SUBJETIVIDADE
DA ANÁLISE
VALIDAÇÃO E
PADRONIZAÇÃO
TUNEL O O O O O O O O O O O
SCSA O O O O O O O O O O O
COMET O O O O O O O O O O O
SCD O O O O O O O
31. Obesidade e Fragmentação DNA
Kort et al, J Androl; 2006
Chavarro et al, Fertil Steril, 2009
19.9
25.8
27
0
5
10
15
20
25
30
IMC 20-24 IMC 25-30 IMC > 30
DFI (%)
IMC 20-24
IMC 25-30
IMC > 30
32. Varicocelectomy studies have reported significant
improvements in one or more semen parameters in
approximately 65% of men
33. Redução média de 14,4% nos IFDNA
Studies have shown that men who achieved a post-operative total motile
sperm count greater than 20 million and sperm DNA fragmentation
decrease after varicocelectomy were more likely to initiate a pregnancy
either spontaneously or via assisted reproductive technology (ART)
34. CASE REPORT
IFDNA DURANTE USO > 30%
3 MESES SEM 21,2 %
APOS 6 MESES 16,5%
ESTUDO
PURAMENTE
ANDROLOGICO
35. Sperm DNA fragmentation after radioiodine
treatment for differentiated thyroid cancer
Background
Treatment of differentiated thyroid cancer usually consists of a total thyroidectomy followed by
one or several courses of radioiodine (131I). 131I is known to have deleterious effects on radiation
sensitive tissues and irradiation to the testes has been shown after its administration. We
investigated effects of such treatment on sperm DNA in a patient with differentiated thyroid
carcinoma.
Methods
The patient, a 32-year-old male with differentiated thyroid carcinoma treated by total
thyroidectomy and radioiodine therapy, performed 6 semen samples in total, 3 for sperm banking
and 3 for semen exploration, that were analysed for classic semen parameters. DNA integrity was
analysed by flow cytometry: sperm DNA fragmentation index (DFI) and high DNA stainability
(HDS) were analyzed by sperm chromatin structure assay, DNA fragmentation was analyzed by
terminal deoxynucleotidyl transferase dUTP nick end labeling assay.
Results
Moderate oligozoospermia was observed as early as 3 months after a first dose of 131I and became
severe at 5 months. Total sperm count was reduced up to 12 months after the second dose of 131I.
Sperm DFI was increased 3.25 months after the first dose of 131I. All parameters returned to
normal values 28 months after the second 131I dose.
36.
37.
38. Radioiodine can affect not only classic
sperm parameters but also the genetic
material of spermatozoa !!!! 2016
Sem análise pré…....
43. 44 pacientes – normo e oligo – SEM CONTROLE
Tratados com zinco (10mg/ml), aspartato (500mg/ml) e
coenzima q10 40mg/ml
Avaliados h/h por 6h e TUNEL ( DNA )
Melhora em todos os parâmetros – motilidade progressiva,
peroxidacao lipidica e DNA FRAGM
44. Can You Treat Sperm DNA Fragmentation
ASRM 2015
Reducing the amount of sperm DNA fragmentation includes such things as
"having a urologist evaluate the man to determine if a varicocele is present
and, if large, surgically repair it. Several studies have shown this not only
brings down the DFI but increases pregnancy success,” .
Keep the testes cool
Stop smoking
Eat a diet rich in antioxidants because oxidative stress is the primary culprit
for breaking DNA
Avoid some medications such as cortisone and SSRIs
Ask your doctor about supplementary vitamins and antioxidants such as
vitamins C and E, co-enzyme Q-10 and supplements containing carnitine.
Fragmentação DNA
45. P < 0,001 após 24hs
Tempo e Temperatura de incubação da amostra seminal
46.
47.
48. Gradiente e Swim up
TÉCNICAS DE PROCESSAMENTO SEMINAL
Swim up
Zini et al
Sakkas et al, Xue et al, Brahem et al
Spano et al, Younglai et al
Swim up e gradiente
49. NOVOS TESTES QUE PERMITAM USAR A CÉLULA AVALIADA
Espectroscopia Raman
50.
51. Fertil Steril. 2015 Dec;104(6):1398-405
ICSI com sptz ejaculado
(N=91)
ICSI com sptz testicular
(N=81)
147 Homens inférteis com oligozoospermia ( 5 – 15 M/mL )
e alta FDE (>30%) após uso de antioxidantes
52. MENSAGENS FINAIS
Espermograma convencional limitado avaliação potencial fértil; resultados devem ser interpretados
com cautela
Teste Fragmentação DNA Espermático emerge como ferramenta que permite avaliação cromatina
nível molecular
No campo laboratorial, as seguintes medidas parecem ser úteis para minimizar a fragmentação do
DNA espermático
b. Processamento seminal pelo gradiente descontínuo coloidal de 2 camadas
c. Incubação por curto período de tempo das amostras pós-processadas (<3h) e à temperatura
ambiente
d. Utilização espermatozoides testiculares
e. Técnicas de seleção espermática (PICSI, Sperm Slow, MACS, IMSI)
Abstinência ejaculatória reduzida (1 dia), com coletas seriadas
Necessário mais estudos (escassos e não padronizados) e meta-análises sobre o tema