This document discusses sperm DNA fragmentation, its importance in fertility evaluation, and techniques for assessing it. Some key points:
- Sperm DNA fragmentation testing provides additional information beyond a standard semen analysis, as up to 20% of infertile men have normal semen parameters but elevated DNA fragmentation.
- Numerous studies since the 2000s have shown increased risks of failed pregnancy and miscarriage associated with high sperm DNA fragmentation levels.
- Techniques for assessing DNA fragmentation include TUNEL, Comet, SCSA, and SCD. SCD is favored for its ease of use.
- Factors like obesity, infections, smoking, varicocele, and certain medications can elevate DNA
11. 16%
Yearly decline in men’s
Sperm count in the West
SOURCE: HUMAN REPRODUCTION UPDATE
12. 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.
13. 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
14. QUAL A IMPORTÂNCIA DO ESTUDO DA
FRAGMENTAÇÃO DA CROMATINA ESPERMÁTICA ?
20% dos homens inférteis apresentam espermograma normal
15. Não é marcador de
fertilidade ou
infertilidade
Até 14% dos espg normais tem
elevados IFDNA
21. 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.
2005
22. 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
23. 2005/6 ---- 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
25. 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:
2007
27. 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
2009
29. 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]
30. 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):
2013
31. 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)
33. Então desde 2012 já se
sabia que quando estava
elevado o casal poderia ter
risco maior de aborto?
34. Críticas até 2018
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
36. 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
44. Varicocelectomy studies have reported significant
improvements in one or more semen parameters in
approximately 65% of men
45. 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)
49. 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.
50.
51. Radioiodine can affect not only classic
sperm parameters but also the genetic
material of spermatozoa !!!! 2016
62. 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
63. 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
66. P < 0,001 após 24hs
Tempo e Temperatura de incubação da amostra seminal
67. 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
69. NOVOS TESTES QUE PERMITAM USAR A CÉLULA AVALIADA
Espectroscopia Raman
70.
71.
72. 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
76. Nunca deixe de solicitar quando
indicado!!!!
Busque sempre a melhora !!!!
Nunca submestime a discussão
intra laboratorial!!!!!
Reze para não ter refratário........
77. MENSAGENS FINAIS
Espermograma convencional limitado avaliação potencial fértil
Teste Fragmentação DNA Espermático ganha força 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 em casos extremos
e. Técnicas de seleção espermática (PICSI)
Abstinência ejaculatória reduzida (1 dia), com coletas seriadas