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A SCSA Diagnostics é pioneira na análise do DNA espermático, utilizando pesquisas consolidadas e estudos clínicos em andamento há mais de 25 anos para realizar um dos testes de fragmentação de DNA espermático mais avançados disponíveis.
O desenvolvimento do teste SCSA foi apresentado na capa da renomada revista Science em dezembro de 1980.
Note que existem alguns laboratórios que oferecem um teste semelhante ao SCSA. No entanto, não é o mesmo teste de precisão da SCSA ®.
A fragmentação do DNA do espermatozoide é uma das principais causas paternas de falha reprodutiva repetida. Cerca de 20-30% dos homens que frequentam uma clínica de fertilidade apresentam um nível de fragmentação do DNA do espermatozoide que pode levar à falha na gravidez..
Spano et al. 2000 A linha tracejada representa o limite clínico para a fragmentação do DNA do espermatozoide e as chances de gravidez.
Alguns autores publicaram que amostras de sêmen que contêm mais de 27-30% de espermatozoides com fragmentações do DNA (% Índice de Fragmentação do DNA) têm de 7 a 10 vezes menor chance de engravidar por meio de relação sexual ou IIU e uma chance 1,5 a 2,0 vezes menor por meio de FIV/ICSI.
Giwercman A et al. (2009, in press, J. Androl) incluíram 137 homens de casais com infertilidade comprovada sem fatores maternos conhecidos e 137 homens com fertilidade comprovada.
Em homens com parâmetros seminais normais (concentração, motilidade e morfologia dos espermatozoides), o risco de infertilidade aumentou 5 vezes com valores de Índice de fragmentação do DNA acima de 20%.
Entretanto, se qualquer um dos parâmetros seminais fosse anormal, o risco de infertilidade aumentava 16 vezes, mesmo com valores de Índice de fragmentação do DNA acima de 10%.
Os autores concluíram que "o Índice de fragmentação do DNA é um preditor da probabilidade de concepção natural, mesmo quando os parâmetros seminais clássicos estão dentro da faixa normal. A análise SCSA complementa o planejamento do tratamento para casais com infertilidade comprovada".
A integridade do DNA espermático é constantemente desafiada por fatores endógenos e exógenos, embora vários mecanismos de reparo e proteção do DNA contra danos estejam ativos nas células humanas (Hoeijmakers, 2009). Em particular, a preservação da integridade do DNA desempenha um papel fundamental na transmissão do genoma para a próxima geração. Nessas células, as quebras de dupla hélice do DNA são induzidas fisiologicamente durante a espermatogênese e a espermiogênese para facilitar o crossing-over meiótico e a substituição de histonas-protaminas, respectivamente (Rathke et al., 2014). Além desses danos "fisiológicos" ao DNA, outros fatores exógenos e endógenos podem afetar a integridade do DNA durante a maturação e o armazenamento dos espermatozoides no epidídimo (Moustafa et al., 2004; Ramos et al., 2004; Sakkas et al., 2002). O estudo da integridade do DNA pode ser um passo fundamental na avaliação da competência funcional dos espermatozoides (Lewis et al., 2008). Portanto, a análise do dano ao DNA espermático pode ser crucial tanto para o diagnóstico de infertilidade quanto para a previsão do sucesso de tecnologias de reprodução assistida. No contexto da FIV, essa avaliação é de particular importância, pois contorna as barreiras naturais de seleção da concepção, aumentando a probabilidade de que os espermatozoides com dano significativo ao DNA transmitam as aberrações genéticas ao recém-nascido (Host et al., 2000). Portanto, vários estudos avaliaram o papel preditivo do dano ao DNA espermático para o resultado das tecnologias de reprodução assistida ou da seleção de espermatozoides.Os danos ao DNA do espermatozoide podem resultar de cinco mecanismos patogênicos diferentes (Perrin et al., 2011; Sakkas e Alvarez, 2010).
IFDNA (Índice de Fragmentação do DNA):
O Índice de Fragmentação do DNA (IFDNA) é a medida da fragmentação do DNA espermático e um indicador da qualidade do espermatozoide.
Células de Alta Coloração de DNA: Porcentagem de espermatozoides com proteínas nucleares anormais e estruturas de cromatina alteradas (espermatozoides imaturos).
O teste SCSA® é realizado usando um dispositivo a laser chamado citômetro de fluxo, que mede 250 espermatozoides por segundo. O resultado do teste é baseado na medição de cerca de 10.000 espermatozoides.
TESTE DE RASTREAMENTO: Especialmente indicado para casais com desejo de ter filhos há muito tempo e sem sucesso
TENTATIVAS MALSUCEDIDAS DE IIU/FIV/ICSI
ABORTOS ESPONTÂNEOS REPETIDOS
BAIXA QUALIDADE E DESENVOLVIMENTO EMBRIONÁRIO
INFERTILIDADE INEXPLICADA: Casais que desejam ter filhos e apresentam testes diagnósticos sem resultados significativos
HOMENS PROPENSOS A ALTO ESTRESSE OXIDATIVO:
ESTILO DE VIDA: tabagismo, uso de drogas, aumento do consumo de álcool, obesidade, estresse
IMPACTO DA NUTRIÇÃO Uma análise detalhada e a modificação apropriada da dieta podem trazer uma melhora drástica na fragmentação do DNA do esperma
EXPOSIÇÃO AO CALOR: por exemplo, banheiras de hidromassagem, trabalhar com um laptop no colo, febre, etc.
EXPOSIÇÕES OCUPACIONAIS: produtos químicos, pesticidas, fertilizantes, corantes, entre outros
DIABETES: Um fator de risco independente para o aumento da fragmentação do DNA espermático
MEDICAMENTOS: Mesmo os medicamentos mais seguros podem causar fragmentação do DNA espermático
TOXINAS AMBIENTAIS / poluição do ar
VARICOCELE: Uma vez que o diagnóstico tenha sido feito, seja por exame clínico ou ultrassom, o teste SCSA pode ajudar a decidir se é necessário fazer a cirurgia ou não
CÂNCER E TRATAMENTOS CONTRA O CÂNCER quimioterapia e radioterapia
DOENÇAS CRÔNICAS: muitas vezes causam aumento do estresse oxidativo, levando a um aumento do dano ao DNA
DOENÇAS ANDROLÓGICAS / Infecções do Trato Geniturinário e Leucospermia: Infecção / inflamação local causa danos diretos ao DNA do espermatozoide
IDADE >40 ANOS: Estudos mostram que quanto mais velho o homem, maior é a fragmentação do DNA do esperma
LUBRIFICANTES: causam danos diretos ao DNA do espermatozoide
O teste SCSA® é realizado usando um dispositivo a laser chamado citômetro de fluxo. Basicamente, o citômetro de fluxo mede 250 espermatozoides/s com alta precisão e fornece um resultado estatisticamente sólido com base em dados de 2x 5000 espermatozoides por amostra. A precisão da citometria de fluxo associada à bioquímica precisa do teste SCSA permite medições altamente precisas da integridade do DNA e da cromatina do esperma.
As células são coradas com um corante de DNA fluorescente (laranja de acridina) e forçadas através de um canal de vidro em suspensão líquida. Quando as células passam pelo feixe de laser, a luz do laser faz com que o corante emita luz fluorescente de uma cor específica..
No teste SCSA®, devido à natureza única do corante Laranja de Acridina, os espermatozoides que emitem fluorescência verde têm níveis indetectáveis de DNA fragmentado, enquanto os espermatozoides que emitem uma fluorescência de amarela à vermelha têm níveis moderados a altos de DNA fragmentado.
Tradicionalmente, a fertilidade masculina é examinada por meio de testes de esperma (espermogramas) que registram a concentração, a motilidade e a morfologia dos espermatozoides. No entanto, numerosos estudos nas últimas cinco décadas demonstram claramente que esses parâmetros não são capazes de prever a gravidez, uma vez que os homens férteis e os inférteis apresentam valores sobrepostos (com exceção da azoospermia). Os espermogramas clássicos são medições microscópicas de fatores externos do esperma, enquanto o teste SCSA mede os fatores internos do núcleo que não podem ser medidos usando o microscópio óptico.
O teste SCSA é altamente preciso, mas requer um citômetro de fluxo que é inacessível para a maioria das clínicas de fertilidade devido aos altos custos de capital e a necessidade de uma equipe treinada. Portanto, as amostras de sêmen coletadas por você ou por um paciente em casa podem ser criopreservadas de forma nativa.
Você pode nos informar por e-mail sobre o transporte desejado. Em seguida, enviaremos a você um recipiente seco pré-resfriado; as amostras de sêmen são armazenadas e enviadas de volta ao nosso laboratório.
O relatório SCSA estará disponível para você via um site seguro aproximadamente 4 dias úteis após a chegada da amostra ao nosso laboratório.
Coleta e processamento das amostras:
A coleta deve ser realizada após 24-48 horas de abstinência, por meio de masturbação em recipientes estéreis de plástico para amostras. Após a liquefação da amostra, a concentração, a motilidade e a morfologia dos espermatozoides são determinadas. Anote os parâmetros no documento de envio.
A concentração dos espermatozoides não deve cair abaixo de 500.000/ml . Caso contrário, podem ocorrer resultados inconclusivos.
A criopreservação deve ocorrer no máximo 1 hora após a entrega , pois o Índice de fragmentação do DNA pode ser alterado após um período de espera mais longo.
Criopreservação das amostras:
As amostras devem ser congeladas o mais rápido possível após a liquefação para minimizar a exposição ao ar, que pode causar danos causados por espécies reativas de oxigênio.
Prepare 2 criotubos (tubos de 1-2 ml): rotule com o nome do paciente e a data da coleta da amostra.
Congelamento rápido :
Pipetar 0,25 ml ou mais do ejaculado nativo liquefeito em cada criotubo.
Não é necessário usar crioprotetores!
Coloque os frascos bem fechados em posição vertical no suporte de metal fornecido e coloque-os diretamente no recipiente criogênico pré-resfriado. O recipiente deve ser fechado rapidamente.
O recipiente é fechado com o lacre fornecido e colocado no recipiente de plástico, ao qual é anexada a nota de envio para que fique claramente visível.
Observação: se a concentração de espermatozoides for suficiente, menos de 0,5 ml (duas alíquotas de 0,25 ml) de ejaculado podem ser suficientes para o teste SCSA®. Para realizar o teste, precisamos de duas alíquotas de 0,20 ml com concentração >0,5M/ml.
Entre em contato com nosso laboratório se tiver alguma dúvida sobre o volume necessário de uma amostra com uma concentração específica.
Envio das amostras:
Nós enviamos um recipiente pré-resfriado. O recipiente armazena o nitrogênio líquido em uma espécie de "esponja" dentro do recipiente, garantindo assim o transporte seguro de suas amostras criopreservadas na fase gasosa de nitrogênio. A baixa temperatura no recipiente pode ser mantida por até 7 dias. Portanto, é importante enviá-lo de volta o mais rápido possível, usando a etiqueta de envio fornecida.
Entre o recipiente e a caixa de envio, há um envelope contendo um lacre e um suporte para ampolas. Também contém uma etiqueta de envio e uma manga correspondente que deve ser anexada ao recipiente para o envio de devolução.
O interior do recipiente é extremamente frio (-196°C). Para evitar queimaduras de gelo, é necessário utilizar luvas ao tocar em qualquer objeto presente no interior do recipiente.
O teste SCSA divide os espermatozoides em cinco populações:
Porcentagem de espermatozoides com cromatina imatura. Os espermatozoides de Células de alta coloração de DNA apresentam menor condensação de cromatina, resultando em maior corante de marcação de DNA. Alguns estudos têm demonstrado que, se a porcentagem de Células de alta coloração de DNA for superior a 36%, nenhuma gravidez ocorreu (Menezo).
Os valores de Índice de fragmentação do DNA (IFDNA) são divididos em quatro categorias estatísticas e podem fornecer as seguintes previsões:
< 15% IFDNA = resultados de gravidez de muito bons a bons
IFDNA de > 15% a < 25% = resultados de gravidez de bons a moderados
IFDNA de > 25% a < 40% = resultados de gravidez de moderados a ruins
IFDNA 40% = resultados de gravidez muito ruins
Todos os dados devem ser compreendidos sem fatores de influência materna.
Além disso, a descoberta mostra uma representação gráfica da porcentagem do Índice de Fragmentação do DNA e da porcentagem de Células de alta coloração de DNA.
O gráfico mostra que 76% dos espermatozoides medidos não apresentam fragmentação do DNA e 24% apresentam fragmentação do DNA de moderada à alta (Índice de fragmentação do DNA = 24%). É possível conceber naturalmente ou com a ajuda de uma inseminação intrauterina (IIU).
Os dados atuais mostram que a probabilidade de uma gravidez bem-sucedida é significativamente menor quando a proporção de espermatozoides com DNA fragmentado é >25%. No entanto, um Índice de fragmentação do DNA >25% não descarta a gravidez. O limite de 25% é um limite estatístico. Se um homem tem um Índice de fragmentação do DNA constante de >25%, ele é colocado em um grupo estatístico que tem mostrado em testes clínicos que leva mais tempo para alcançar a gravidez naturalmente e requer mais ciclos de fertilização in vitro (FIV). Uma taxa aumentada de abortos espontâneos está associada a isso ou a gravidez não ocorre de forma alguma.
Avaliando a qualidade do DNA do espermatozoide
Se o Índice de fragmentação do DNA e as Células de alta coloração de DNA estão dentro da faixa normal, a gravidez é possível naturalmente ou por inseminação (ignorando fatores do parceiro que afetam a fertilidade).
Isso significa que o DNA do espermatozoide apresenta uma alta estabilidade.
O Índice de fragmentação do DNA está significativamente aumentado. Isso significa que o DNA do espermatozoide é muito frágil e tende à fragmentação do DNA.
A estabilização do DNA pode ser alcançada por meio de:
Em alguns casos, pode ser necessário mudar de IIU para FIV/ICSI para aumentar as chances de um resultado positivo de gravidez.
The histogram shows the DFI (DNA Fragmentation Index) measured in a semen sample.
DFI scores are classified into four statistical categories and can predict the following (ignoring partner factors affecting fertility):
< 15% DFI = Very good to good pregnancy outcomes
> 15% to < 25% DFI = Good to moderate pregnancy outcomes
> > 25% to < 40% DFI = Moderate to poor pregnancy outcomes
> 40% DFI = Very bad outcome of pregnancy
If the %DFI is above 25%, the current literature recommends:
Acredita-se que o óvulo seja capaz de reparar certos níveis de danos no DNA.
No entanto, os níveis de moderados a altos de fragmentação do DNA (Índice de fragmentação do DNA) encontrados nos espermatozoides fluorescentes vermelhos provavelmente excedem a capacidade de reparação do DNA do óvulo.
Marcos Mesequer et al. descobriram que após a recuperação do óvulo, cada aumento de 10% no Índice de fragmentação do DNA aumentou em 1,31 vezes as chances de não engravidar. Em comparação, esse efeito não foi observado quando se utilizou óvulos doados, o que indica uma maior capacidade de reparação com relação à fragmentação do DNA de óvulos jovens.
Se o Índice de fragmentação do DNA for alto, recomendamos:
Se houver um fator de risco, é recomendado repetir o teste SCSA 3 meses após a remoção/correção do fator de risco para avaliar o impacto.
Exemplos:
Abstinência de nicotina (84 dias depois):
Cirurgia de varicocele (3 meses depois):
Cessação da exposição a pesticidas (3 meses depois):
O custo do teste é de €295. Atualmente, não é coberto pelos planos de saúde.
O envio das amostras ao nosso laboratório é gratuito.
Para valores de Índice de fragmentação do DNA >30%, oferecemos um segundo teste de controle gratuito, que normalmente deve ser realizado 3 meses após o primeiro teste.
Fragmentation of sperm DNA is one of the most important paternal causes of repeated reproductive failure. About 20-30% of men who attend a fertility clinic have a level of sperm DNA fragmentation that can lead to pregnancy failure.
Spano et al. 2000 The dashed line shows the clinical threshold for sperm DNA fragmentation and pregnancy chances
Some authors have published that semen samples containing more than 27-30% sperm with DNA fragmentations (DNA Fragmentation Index, %DFI) are 7-10 times less likely to get pregnant through intercourse or IUI, and one around 1.5 -2.0 times lower chance through IVF/ICSI
Giwercman A et al. (2009 in press, Int. J. Androl) included 137 men from couples with proven infertility with no known maternal factors and 137 men with proven fertility
In men with normal semen parameters (sperm concentration, motility and morphology), the risk of infertility was increased 5-fold with DFI values above 20%.
However, if any of the semen parameters were abnormal, the risk of infertility was increased 16-fold even with DFI values above 10%
The authors concluded that “DFI is a predictor of the likelihood of natural conception, even when classic semen parameters are within the normal range. The SCSA analysis complements treatment planning for couples with proven infertility.”
Sperm DNA integrity is continuously challenged by endogenous and exogenous factors, although different mechanisms of repairing and protecting against this damage are active in human cells (Hoeijmakers, 2009). This is particularly relevant in germ cells, which have to preserve DNA integrity to pass the genome to the next generation. In these cells, DNA double-strand breaks are physiologically induced during spermatogenesis and spermiogenesis to facilitate meiotic crossover and histone– protamine substitution, respectively (Rathke et al., 2014). Apart from this first ‘physiological’ DNA damage, other exogenous and endogenous factors could affect DNA integrity during sperm maturation and storage in the epididymis (Moustafa et al., 2004; Ramos et al., 2004; Sakkas et al., 2002). Thus, DNA integrity is constantly at risk and its assessment could be a fundamental step in the evaluation of sperm functional competence (Lewis et al., 2008). Hence, sperm DNA damage evaluation could be crucial for both infertility diagnosis and prediction of ART success. In the setting of IVF this evaluation plays a peculiar role because the natural selection barriers of conception are bypassed, increasing the possibility of spermatozoa with significant DNA damage transmitting the genetic aberrations to the newborn (Host et al., 2000). Thus, several trials have evaluated the predictive role of sperm DNA damage for either ART outcome or sperm selection.
DFI (DNA Fragmentation Index) :
Percentage of sperm with DNA fragmentation
HDS (High DNA Stainability Cells): Percentage of sperm with abnormal core proteins and chromatin structures (immature sperm)
The SCSA® test is performed using a laser-based device called a flow-cytometer which measures 250 sperm/second. The test result is based on the measurment of around 10,000 sperm.
LIFESTYLE: smoking, drug use, increased alcohol consumption, obesity, stress
NUTRITION: Although commonly overlooked, we have often seen drastic improvement in sperm DNA fragmentation after a detailed analysis and correction of daily nutrition.
HEAT EXPOSURE: e.g. from whirlpool baths, working with a laptop on the lap, fever, etc.
OCCUPATIONAL EXPOSURES: chemicals, pesticides, fertilizers, dyes, etc.
DIABETES: An independent risk factor for increased sperm DNA fragmentation
MEDICATION: Even the most unassuming medications have sometimes seen to cause sperm DNA fragmentation
ENVIRONMENTAL TOXINS /air pollution: Although it may appear general, a keen look into exposure should be ruled out.
VARICOCELE: Once diagnosed either by examination or ultrasound, SCSA test can help in the decision if surgery will help.
CANCER AND CANCER TREATMENTS (chemotherapy and radiotherapy) : causes direct damage to the DNA of cells.
CHRONIC DISEASES: often cause increased oxidative stress leading to increased DNA damage
ANDROLOGICAL DISEASES / Infections of the urogenital tract and leukocytospermia: Local infection / inflammation causes direct damage to the DNA in sperm through release of ROS (Reactive Oxygen Species).
AGE >40 YEARS: It has been extensively studied that the older the man, the higher the DNA fragmentation
LUBRICANTS: cause direct damage to the sperm DNA
Sperm DNA damage can result from five different pathogenic mechanisms (Perrin et al., 2011; Sakkas and Alvarez, 2010).
The SCSA® test is performed using a laser-based device called a flow cytometer. Essentially, the flow cytometer measures 250 sperm/s with high precision, providing a statistically robust result based on data from 2x 5000 sperm per sample. The precision of flow cytometry coupled with the precise biochemistry of the SCSA test provides highly accurate measurements of sperm chromatin and DNA integrity.
The cells are stained with a fluorescent DNA stain (acridine orange) and then forced through a glass channel in a liquid suspension. When the cells pass the laser beam, the laser light causes the dye to emit fluorescent light of a specific color.
In the SCSA® test, due to the unique nature of the Acridine Orange dye, sperm that emit green fluorescence have undetectable levels of fragmented DNA, while sperm that emit yellow to red florescence have moderate to high levels of fragmented DNA.
The SCSA test is a two-fold simultaneous flow cytometry measure of
in thousands of sperm in a fresh or frozen/thawed semen sample.
The extent of DNA strand breaks and abnormality of chromatin structure are related to male factor fertility, including time to couple pregnancy, IVF embryo quality, miscarriage, or infertility.
Male fertility is classically addressed by semen tests that include sperm density, motility and morphology. However, numerous studies over the past half century clearly show that, except for absence of sperm, these parameters do not predict pregnancy since fertile and infertile men have overlapping values. The classical semen tests are light microscope measures of external sperm factors while the SCSA test are measures of the internal nuclear factors not visible by light
microscopy.
The SCSA test is a high precision test that requires a flow cytometer which due to the need of a high capital expense and a trained technician is not amenable to most infertility clinics. Thus, semen samples collected, as per detailed instructions, in a clinic or in a patients’ home, can be flash frozen in a LN 2 dry shipper tank.(See support protocol 1).
Clinics can then send us an email informing us. A pre-cooled LN 2 shipper is then sent to the clinic and the semen samples are flash frozen and the LN 2 shipper then returned to a SCSA diagnostic testing lab.
Some clinics send frozen semen samples in well insulated dry ice containers.
SCSA clinical data are then sent via a secure WEB site to the physician or patient that ordered the test.
There are five main populations of sperm identified by the SCSA® test:
the percentage of sperm with immature chromatin. HDS sperm exhibit less chromatin condensation, resulting in increased DNA stainability. Some studies have shown that if the %HDS is above 36%, no pregnancies have occurred (Menezo)
Our SCSA clinical report lists four statistical categories of fertility potential. These were derived from a comprehensive study of male fertility potential without medical intervention and have been confirmed in additional studies.
< 15% DFI = Excellent to good pregnancy outcomes (excluding female infertility factors)
> 15% to < 25% DFI = good to fair pregnancy outcomes
> 25% to < 40% DFI = fair to poor pregnancy outcomes
> 40% DFI = Very poor pregnancy outcomes
Furthermore, the finding shows a graphical representation of the %DFI (DNA Fragmentation Index) and the %HDS (high DNA stainability)
The graph shows that 76% of the measured sperm shows no DNA fragmentation and 24% shows medium-high DNA fragmentation (DFI= 24%). Pregnancy naturally or with the help of an IUI is possible.
Current data shows that the probability of a successful pregnancy is significantly lower when the proportion of sperm with fragmented DNA is > 25%. Nevertheless, a DFI > 25% does not rule out a normal pregnancy. The 25% threshold is a statistical threshold. Therefore, if a man has a constant DFI >25%, he will be placed in a statistical group of men who have been shown in clinical trials to take longer to achieve natural pregnancy and more IVF cycles. An increased rate of spontaneous abortions is associated with this or pregnancy does not occur at all.
The ovum is believed to be able to repair a certain amount of DNA damage.
Moderate to high levels of DNA fragmentation (DFI) found in the red fluorescent sperm likely exceed the egg’s DNA repair capacity.
Marcos Meseguer et al found that when oocytes from patients were taken, for every 10% increase in DFI, there was a 1.31 times increase in probability of not achieving pregnancy. Whereas, the same was not seen when donor oocytes were employed suggesting the higher capability of young oocytes to repair sperm DNA damage.
REPEATED IUI / IVF / ICSI FAILURE : The reason behind the unsuccessful attempts at IUI/IVF/ICSI can be identified and managed
REPEATED MISCARRAGE : An explanation for repeated spontaneous abortions (RPL, repeated miscarriage) could be found and guided
POOR EMBRYO DEVELOPMENT AND QUALITY : The reason behind poor embryo quality could be the fragmented sperm DNA which could be corrected before the next treatment
MEN AT RISK FOR HIGHER OXIDATIVE STRESS:INITIAL SCREENING TEST : Especially for couples who have been attempting pregnancy for a long time to identify the best management strategy
UNEXPLAINED INFERTILITY : Couples desiring children with all normal diagnostic tests
In cases of high DFI, we suggest:
When a risk factor is identified, after avoidance / correction of the risk factor a repeat SCSA test after 3 months is advised.
Yes. We offer a SCSA test at half the cost for controlling the effect of avoidance / correction of a risk factor.
Please find a few examples below:
After quitting smoking for 84 days
3 months after varicocele correction surgery :
3 months after stopping pesticide exposure:
After 24-48 hours of abstinence, the samples should be taken by masturbation into sterile plastic sample containers. After liquefaction of the sample, the sperm concentration, motility and morphology are determined.
Cryopreservation should take place no more than 1 hour after delivery, since the DFI can be falsely raised after a longer waiting period.
Flash-freezing: Freeze 0.25mL or more of liquefied native ejaculate in 1-2mL cryovials. Cryoprotectants is not required! (Please freeze 2 cryovials per patient)
Samples should be frozen as soon as possible after liquefaction to minimize exposure to air, which can cause damage caused by reactive oxygen species.Cryovials should be tightly capped, labeled with two identifiers (e.g. name and date of birth) and placed in an upright position directly into the pre-chilled cryo-container. The container must be closed quickly.
Note: Cryoprotectants are not needed since quick-frozen sperm give the clearest SCSA® data. This feature is unique to mammalian sperm cells due to the highly condensed, crystalline nature of the nuclei.
Note: If the sperm concentration is adequate, less than 0.5 mL (two 0.25 mL aliquots) of semen may be sufficient for the SCSA® test. To perform the test, we require two 0.20 mL aliquots with a concentration of >0.5 million/ml. Please contact our laboratory if you have questions pertaining to the volume necessary for a sample of a certain concentration.
We ship a pre-chilled dry shipper. Dry shippers store the liquid nitrogen in porous material and thus ensure the safe transport of your cryopreserved samples in the nitrogen gas phase.
The low temperature in the container can be maintained for 7 days. It is therefore important to send it back as soon as possible using the mailed/supplied shipping label.
Note: liquid nitrogen is adsorbed by special materials inside the shipping container. Even if they are turned upside down during transport, no liquid nitrogen will leak out. Dry shippers are not considered dangerous.
After receiving the sample(s) in our laboratory, we need 4 working days to create a report, which we will send to you.
The cost of the test is 295 Euro.
* Samples are shipped in a pre-cooled shipper that we can provide either at ambient or pre-chilled temperatures. The pre-cooled shipper will be sent to the centre where you have provided your sample for the centre to ship it to us.
The test at present is not covered by the insurance.
Please write us an email to contact@scsalab.com and after contacting you, we will send toy a dry shipper for the patient sample to be sent to us.
Fragmentation of sperm DNA is one of the most important paternal causes of repeated reproductive failure. About 20-30% of men who attend a fertility clinic have a level of sperm DNA fragmentation that can lead to pregnancy failure.
Spano et al. 2000 The dashed line shows the clinical threshold for sperm DNA fragmentation and pregnancy chances
Some authors have published that semen samples containing more than 27-30% sperm with DNA fragmentations (DNA Fragmentation Index, %DFI) are 7-10 times less likely to get pregnant through intercourse or IUI, and one around 1.5 -2.0 times lower chance through IVF/ICSI
Giwercman A et al. (2009 in press, Int. J. Androl) included 137 men from couples with proven infertility with no known maternal factors and 137 men with proven fertility
In men with normal semen parameters (sperm concentration, motility and morphology), the risk of infertility was increased 5-fold with DFI values above 20%.
However, if any of the semen parameters were abnormal, the risk of infertility was increased 16-fold even with DFI values above 10%
The authors concluded that “DFI is a predictor of the likelihood of natural conception, even when classic semen parameters are within the normal range. The SCSA analysis complements treatment planning for couples with proven infertility.”
Sperm DNA integrity is continuously challenged by endogenous and exogenous factors, although different mechanisms of repairing and protecting against this damage are active in human cells (Hoeijmakers, 2009). This is particularly relevant in germ cells, which have to preserve DNA integrity to pass the genome to the next generation. In these cells, DNA double-strand breaks are physiologically induced during spermatogenesis and spermiogenesis to facilitate meiotic crossover and histone– protamine substitution, respectively (Rathke et al., 2014). Apart from this first ‘physiological’ DNA damage, other exogenous and endogenous factors could affect DNA integrity during sperm maturation and storage in the epididymis (Moustafa et al., 2004; Ramos et al., 2004; Sakkas et al., 2002). Thus, DNA integrity is constantly at risk and its assessment could be a fundamental step in the evaluation of sperm functional competence (Lewis et al., 2008). Hence, sperm DNA damage evaluation could be crucial for both infertility diagnosis and prediction of ART success. In the setting of IVF this evaluation plays a peculiar role because the natural selection barriers of conception are bypassed, increasing the possibility of spermatozoa with significant DNA damage transmitting the genetic aberrations to the newborn (Host et al., 2000). Thus, several trials have evaluated the predictive role of sperm DNA damage for either ART outcome or sperm selection.
DFI (DNA Fragmentation Index) :
Percentage of sperm with DNA fragmentation
HDS (High DNA Stainability Cells): Percentage of sperm with abnormal core proteins and chromatin structures (immature sperm)
The SCSA® test is performed using a laser-based device called a flow-cytometer which measures 250 sperm/second. The test result is based on the measurment of around 10,000 sperm.
LIFESTYLE: smoking, drug use, increased alcohol consumption, obesity, stress
NUTRITION: Although commonly overlooked, we have often seen drastic improvement in sperm DNA fragmentation after a detailed analysis and correction of daily nutrition.
HEAT EXPOSURE: e.g. from whirlpool baths, working with a laptop on the lap, fever, etc.
OCCUPATIONAL EXPOSURES: chemicals, pesticides, fertilizers, dyes, etc.
DIABETES: An independent risk factor for increased sperm DNA fragmentation
MEDICATION: Even the most unassuming medications have sometimes seen to cause sperm DNA fragmentation
ENVIRONMENTAL TOXINS /air pollution: Although it may appear general, a keen look into exposure should be ruled out.
VARICOCELE: Once diagnosed either by examination or ultrasound, SCSA test can help in the decision if surgery will help.
CANCER AND CANCER TREATMENTS (chemotherapy and radiotherapy) : causes direct damage to the DNA of cells.
CHRONIC DISEASES: often cause increased oxidative stress leading to increased DNA damage
ANDROLOGICAL DISEASES / Infections of the urogenital tract and leukocytospermia: Local infection / inflammation causes direct damage to the DNA in sperm through release of ROS (Reactive Oxygen Species).
AGE >40 YEARS: It has been extensively studied that the older the man, the higher the DNA fragmentation
LUBRICANTS: cause direct damage to the sperm DNA
Sperm DNA damage can result from five different pathogenic mechanisms (Perrin et al., 2011; Sakkas and Alvarez, 2010).
The SCSA® test is performed using a laser-based device called a flow cytometer. Essentially, the flow cytometer measures 250 sperm/s with high precision, providing a statistically robust result based on data from 2x 5000 sperm per sample. The precision of flow cytometry coupled with the precise biochemistry of the SCSA test provides highly accurate measurements of sperm chromatin and DNA integrity.
The cells are stained with a fluorescent DNA stain (acridine orange) and then forced through a glass channel in a liquid suspension. When the cells pass the laser beam, the laser light causes the dye to emit fluorescent light of a specific color.
In the SCSA® test, due to the unique nature of the Acridine Orange dye, sperm that emit green fluorescence have undetectable levels of fragmented DNA, while sperm that emit yellow to red florescence have moderate to high levels of fragmented DNA.
The SCSA test is a two-fold simultaneous flow cytometry measure of
in thousands of sperm in a fresh or frozen/thawed semen sample.
The extent of DNA strand breaks and abnormality of chromatin structure are related to male factor fertility, including time to couple pregnancy, IVF embryo quality, miscarriage, or infertility.
Male fertility is classically addressed by semen tests that include sperm density, motility and morphology. However, numerous studies over the past half century clearly show that, except for absence of sperm, these parameters do not predict pregnancy since fertile and infertile men have overlapping values. The classical semen tests are light microscope measures of external sperm factors while the SCSA test are measures of the internal nuclear factors not visible by light
microscopy.
The SCSA test is a high precision test that requires a flow cytometer which due to the need of a high capital expense and a trained technician is not amenable to most infertility clinics. Thus, semen samples collected, as per detailed instructions, in a clinic or in a patients’ home, can be flash frozen in a LN 2 dry shipper tank.(See support protocol 1).
Clinics can then send us an email informing us. A pre-cooled LN 2 shipper is then sent to the clinic and the semen samples are flash frozen and the LN 2 shipper then returned to a SCSA diagnostic testing lab.
Some clinics send frozen semen samples in well insulated dry ice containers.
SCSA clinical data are then sent via a secure WEB site to the physician or patient that ordered the test.
There are five main populations of sperm identified by the SCSA® test:
the percentage of sperm with immature chromatin. HDS sperm exhibit less chromatin condensation, resulting in increased DNA stainability. Some studies have shown that if the %HDS is above 36%, no pregnancies have occurred (Menezo)
Our SCSA clinical report lists four statistical categories of fertility potential. These were derived from a comprehensive study of male fertility potential without medical intervention and have been confirmed in additional studies.
< 15% DFI = Excellent to good pregnancy outcomes (excluding female infertility factors)
> 15% to < 25% DFI = good to fair pregnancy outcomes
> 25% to < 40% DFI = fair to poor pregnancy outcomes
> 40% DFI = Very poor pregnancy outcomes
Furthermore, the finding shows a graphical representation of the %DFI (DNA Fragmentation Index) and the %HDS (high DNA stainability)
The graph shows that 76% of the measured sperm shows no DNA fragmentation and 24% shows medium-high DNA fragmentation (DFI= 24%). Pregnancy naturally or with the help of an IUI is possible.
Current data shows that the probability of a successful pregnancy is significantly lower when the proportion of sperm with fragmented DNA is > 25%. Nevertheless, a DFI > 25% does not rule out a normal pregnancy. The 25% threshold is a statistical threshold. Therefore, if a man has a constant DFI >25%, he will be placed in a statistical group of men who have been shown in clinical trials to take longer to achieve natural pregnancy and more IVF cycles. An increased rate of spontaneous abortions is associated with this or pregnancy does not occur at all.
The ovum is believed to be able to repair a certain amount of DNA damage.
Moderate to high levels of DNA fragmentation (DFI) found in the red fluorescent sperm likely exceed the egg’s DNA repair capacity.
Marcos Meseguer et al found that when oocytes from patients were taken, for every 10% increase in DFI, there was a 1.31 times increase in probability of not achieving pregnancy. Whereas, the same was not seen when donor oocytes were employed suggesting the higher capability of young oocytes to repair sperm DNA damage.
REPEATED IUI / IVF / ICSI FAILURE : The reason behind the unsuccessful attempts at IUI/IVF/ICSI can be identified and managed
REPEATED MISCARRAGE : An explanation for repeated spontaneous abortions (RPL, repeated miscarriage) could be found and guided
POOR EMBRYO DEVELOPMENT AND QUALITY : The reason behind poor embryo quality could be the fragmented sperm DNA which could be corrected before the next treatment
MEN AT RISK FOR HIGHER OXIDATIVE STRESS:INITIAL SCREENING TEST : Especially for couples who have been attempting pregnancy for a long time to identify the best management strategy
UNEXPLAINED INFERTILITY : Couples desiring children with all normal diagnostic tests
In cases of high DFI, we suggest:
When a risk factor is identified, after avoidance / correction of the risk factor a repeat SCSA test after 3 months is advised.
Yes. We offer a SCSA test at half the cost for controlling the effect of avoidance / correction of a risk factor.
Please find a few examples below:
After quitting smoking for 84 days
3 months after varicocele correction surgery :
3 months after stopping pesticide exposure:
After 24-48 hours of abstinence, the samples should be taken by masturbation into sterile plastic sample containers. After liquefaction of the sample, the sperm concentration, motility and morphology are determined.
Cryopreservation should take place no more than 1 hour after delivery, since the DFI can be falsely raised after a longer waiting period.
Flash-freezing: Freeze 0.25mL or more of liquefied native ejaculate in 1-2mL cryovials. Cryoprotectants is not required! (Please freeze 2 cryovials per patient)
Samples should be frozen as soon as possible after liquefaction to minimize exposure to air, which can cause damage caused by reactive oxygen species.Cryovials should be tightly capped, labeled with two identifiers (e.g. name and date of birth) and placed in an upright position directly into the pre-chilled cryo-container. The container must be closed quickly.
Note: Cryoprotectants are not needed since quick-frozen sperm give the clearest SCSA® data. This feature is unique to mammalian sperm cells due to the highly condensed, crystalline nature of the nuclei.
Note: If the sperm concentration is adequate, less than 0.5 mL (two 0.25 mL aliquots) of semen may be sufficient for the SCSA® test. To perform the test, we require two 0.20 mL aliquots with a concentration of >0.5 million/ml. Please contact our laboratory if you have questions pertaining to the volume necessary for a sample of a certain concentration.
We ship a pre-chilled dry shipper. Dry shippers store the liquid nitrogen in porous material and thus ensure the safe transport of your cryopreserved samples in the nitrogen gas phase.
The low temperature in the container can be maintained for 7 days. It is therefore important to send it back as soon as possible using the mailed/supplied shipping label.
Note: liquid nitrogen is adsorbed by special materials inside the shipping container. Even if they are turned upside down during transport, no liquid nitrogen will leak out. Dry shippers are not considered dangerous.
After receiving the sample(s) in our laboratory, we need 4 working days to create a report, which we will send to you.
The cost of the test is 295 Euro.
* Samples are shipped in a pre-cooled shipper that we can provide either at ambient or pre-chilled temperatures. The pre-cooled shipper will be sent to the centre where you have provided your sample for the centre to ship it to us.
The test at present is not covered by the insurance.
Please write us an email to contact@scsalab.com and after contacting you, we will send toy a dry shipper for the patient sample to be sent to us.