WORKSHOP BOOK Fluorescent in Situ Hybridisation - FISH - on Human Sperm

1 

<strong>WORKSHOP</strong> <strong>BOOK</strong> 

<strong>Fluorescent</strong> <strong>in</strong> <strong>Situ</strong> <strong>Hybridisation</strong> - <strong>FISH</strong> 

- on Human Sperm 

31.08.2014

2 

Hands-on workshops <strong>in</strong> specialised 

ART laboratory techniques 

Thessaloniki, Greece 

August 31 – September 5, 2014 

Dear Embryologist, 

Dear Scientist, 

Dear Dr, 

Dear Colleague, 

Welcome to Thessaloniki, Greece! 

Welcome to the Embryolab Academy ‘Hands-on Workshops <strong>in</strong> specialised ART laboratory 

techniques’! 

You will be tak<strong>in</strong>g part <strong>in</strong> one (or more) of the 5 different Hands-on Workshops <strong>in</strong> specialised ART 

laboratory techniques. 

Embryolab Academy has prepared a busy schedule for you: theoretical morn<strong>in</strong>g lectures will give an 

overview of recent advances for each technique. Afternoon sessions are entirely devoted to 

<strong>in</strong>dividualised Hands-on tra<strong>in</strong><strong>in</strong>g under guidance of your expert teachers. 

We hope you will enjoy your tra<strong>in</strong><strong>in</strong>g as well as your stay <strong>in</strong> Thessaloniki! 

K<strong>in</strong>d regards 

Workshop Organisers 

Mart<strong>in</strong>e Nijs | Alexia Chatziparasidou | Nikos Christoforidis

3 

Content Workshop Book 

Embryolab Academy 

Overview 

4 

Hands-on workshops <strong>in</strong> specialised ART laboratory techniques 

Workshop 1 description 

5 

<strong>Fluorescent</strong> <strong>in</strong> <strong>Situ</strong> <strong>Hybridisation</strong> - <strong>FISH</strong> - on Human Sperm 

Expert Teachers 6 

Programme Workshop 1 7 

Lectures 

Aneuploidy <strong>in</strong> male <strong>in</strong>fertile patients: <strong>in</strong>dications and results. 

8 

Alexia Chatziparasidou 

<strong>FISH</strong> on human sperm: technical aspects. 

34 

Glykeria Samolada 

<strong>FISH</strong> technique: Standard Operat<strong>in</strong>g procedure, materials and methods, 

report<strong>in</strong>g, Internal and external quality control for <strong>FISH</strong>. 

55 


<strong>FISH</strong> SOP Embryolab 63 

Participants list 70 

Our generous sponsors 71 

General <strong>in</strong>formation 72 

About Embryolab Academy 74

4 

Hands-on workshops <strong>in</strong> specialised ART 

laboratory techniques 

August 31 st - September 5-9-2014 

Workshop 

number 

Hands-on Workshop 

Date 

1 <strong>Fluorescent</strong> <strong>in</strong> <strong>Situ</strong> Hybridization - <strong>FISH</strong> - on Human Sperm 31.08.2014 

2 Quality and Risk Management <strong>in</strong> IVF Laboratories 01.09.2014 

3 Vitrification of oocytes, cleavage stage embryos and blastocysts 02.09.2014 

4 Biopsy of polar bodies, blastomeres, trophectoderm cells - 

Preparation of material for genetic analysis - Tub<strong>in</strong>g of cells* 

03-04.09 2014 

5 Intra cytoplasmic sperm <strong>in</strong>jection - ICSI 05.09.2014 

* Advanced course, admission accepted after submission of short CV with track record <strong>in</strong> micromanipulation

5 

Hands-on Workshop 1 

<strong>Fluorescent</strong> <strong>in</strong> <strong>Situ</strong> <strong>Hybridisation</strong> - 

<strong>FISH</strong> - on Human Sperm 

Date: 31.08.2014 

Venue: Embryolab Academy 

173-175 Ethnikis Antistaseos, 551 34 Kalamaria, Thessaloniki, Greece 

Maximum number of participants: 5 

Aim and goals of the course: 

Understand importance of diagnosis of aneuploidy <strong>in</strong> male patients. 

Explore current sperm aneuploidy assessment techniques. 

Quality control and efficient report<strong>in</strong>g of outcomes. 

Have Hands-on tra<strong>in</strong><strong>in</strong>g <strong>in</strong> <strong>FISH</strong> on sperm. 

Target Audience 

Embryolab Academy Hands-on Workshops are designed for reproductive scientists, 

embryologists, lab technicians, geneticists and cl<strong>in</strong>icians who desire to improve their knowledge 

and practical skills <strong>in</strong> specialised laboratory techniques used <strong>in</strong> a human ART sett<strong>in</strong>g.

6 

Expert teachers 

Glykeria Salmolada 

M Sc, Bac Sc, Molecular Cytogeneticist 

Associate Geneticists Embryolab, Thessaloniki, Greece. 

Mrs. Glykeria Samolada is a biologist specialised <strong>in</strong> Molecular Cytogenetics. 

Glykeria has a vast experience <strong>in</strong> research as well as the cl<strong>in</strong>ical application of 

molecular techniques <strong>in</strong> the field of cancer monitor<strong>in</strong>g and diagnosis. Mrs. Glykeria Samolada 

graduated from the Biology Department of Aristotle University of Thessaloniki <strong>in</strong> 1992. In 2005 

she obta<strong>in</strong>ed her post-Graduate Diploma <strong>in</strong> Molecular Cytogenetics at the Université de 

Montpellier. 

S<strong>in</strong>ce 2005 she practises different techniques of conventional karyotyp<strong>in</strong>g, <strong>FISH</strong>, M-<strong>FISH</strong> and 

CGH. She followed an Internship at the Molecular Cytogenetics Lab of the Institut für Human 

Genetics, Univ. Kl<strong>in</strong>ikum Heidelberg and <strong>in</strong> the Laboratory of Cytogenetics of the Haematology 

Cl<strong>in</strong>ic of the Papanikolaou University Hospital. Glykeria is Head of INVIVO Biomedical Institute 

and s<strong>in</strong>ce 2009 she is an Associate Geneticist at Embryolab. 

She is a Member of the European Cytogenetic Association (ECA) and obta<strong>in</strong>ed a Certificate on 

Health Sciences and Genetic Counsell<strong>in</strong>g (2014, Un Plymouth). 


M Cl<strong>in</strong> Embryology, M Sc, Bac Sc, Sr Cl<strong>in</strong>ical Embryologist 

Laboratory Director Embryolab, Thessaloniki, Greece; h-<strong>in</strong>dex: 3 

Mrs. Alexia Chatziparasidou is a Sr. Cl<strong>in</strong>ical Embryologist with long 

experience of more than 30,000 IVF cycles. Her scientific <strong>in</strong>terests focus 

ma<strong>in</strong>ly on the application of modern fertility preservation techniques. She 

has also long experience on treat<strong>in</strong>g male subfertility, as well as on develop<strong>in</strong>g methods of coculture 

with homologous endometrial cells. 

In 2007, Mrs Chatziparasidou completed her M Sc <strong>in</strong> Cl<strong>in</strong>ical Embryology at the University of 

Leeds, UK. S<strong>in</strong>ce 2007 she is actively <strong>in</strong>volved <strong>in</strong> preimplantation genetic diagnosis and 

screen<strong>in</strong>g <strong>in</strong> couples at risk. In 2004, Mrs Chatziparasidou founded Embryolab, and s<strong>in</strong>ce has 

been the director of Embryolab, a modern state of art assisted reproduction unit. 

She is member of Alpha, the <strong>in</strong>ternational society of Reproductive Scientists , the European 

Society of Human Reproduction and the American Society of Reproductive Medic<strong>in</strong>e. Mrs 

Chatziparasidou is co-founder of Embryolab-Academy.

7 

Programme 

8:30 – 8:45 Pick- up from hotel – transportation to Embryolab Academy 

9:00 – 9:15 

9:15 – 10:00 

10:00 – 10:45 

Welcome by the Director of Embryolab Academy 

Mart<strong>in</strong>e Nijs 

Aneuploidy <strong>in</strong> male <strong>in</strong>fertile patients: <strong>in</strong>dications and results. 


<strong>FISH</strong> on human sperm: technical aspects. 


10:45 –11:00 Coffee break 

11:00 – 12:30 

<strong>FISH</strong> technique: Standard Operat<strong>in</strong>g procedure, materials and methods, 

report<strong>in</strong>g, Internal and external quality control for <strong>FISH</strong>. 


12:30 Lunch 

13:15 – 17:30 

17:30 – 17:45 

Hands-on <strong>in</strong>clud<strong>in</strong>g 


Fixation, hybridisation and microscopic analysis of <strong>FISH</strong> on human 

sperm 

Troubleshoot<strong>in</strong>g <strong>in</strong> <strong>FISH</strong> on sperm 

Summary of workshop 

Mart<strong>in</strong>e Nijs 

17:45 – 18:00 CPD - Award<strong>in</strong>g of certificates - Farewell 

18:00 Return to hotel

8 

Aneuploidy <strong>in</strong> male <strong>in</strong>fertile patients: 

<strong>in</strong>dications and results 

Alexia Chatziparasidou

Aneuploidy <strong>in</strong> male <strong>in</strong>fertile patients: 

Indications and results 

Alexia Chatziparasidou, MSc 

1 

Declaration of Interest 

Conflict of <strong>in</strong>terest: 

Lab Director of Embryolab 

Hands-on Workshops, Thessaloniki, 30.08 – 05.09 2014 

2

Aneuploidy <strong>in</strong> male <strong>in</strong>fertile patients 

General population 

Males with abnormal karyotypes 

Infertile males with normal karyotypes 

Males exposed to certa<strong>in</strong> environmental/life style hazards 

Risk reduction 

Future 


3 

4

Spermatogenesis 


5 

Why is it important to assess the risk? 

With the advent of assisted reproduction 

technologies we can overcome many types of male 

<strong>in</strong>fertility and hence bypass nature’s protective 

mechanisms that were developed <strong>in</strong> evolution to 

prevent fertilization with a defective or deficient 

sperm. 

Hwang et al, 2010 


6

Multicolor sperm <strong>FISH</strong> 

(Fluorescence In <strong>Situ</strong> Hybridization) 


7 

Sperm aneuploidy 

<strong>in</strong> general male population 

a disomy rate of 

0.1% and a total 

aneuploidy of 4.5% 

Templado et al, 2011 


8

Paternally derived aneuploidies 

A 2x <strong>in</strong>crease <strong>in</strong> disomy frequency for chromosomes 21 

and XY <strong>in</strong> fathers of paternally derived Down, Turner and 

Kl<strong>in</strong>efelter syndromes. 

Arnedo et al, 2006; Templado et al, 2011 

A 2.3x <strong>in</strong>crease <strong>in</strong> sex chromosome disomy frequency <strong>in</strong> 

men with repeated spontaneous abortions 

Collodel et al, 2009; Robio et al, 1999 


9 

Conclusion: General Population 

In the general population the estimated total paternally 

derived aneuploidy risk is low. 

However, among fertile men, some may consistently 

produce higher levels of aneuploidy (stable variants) at least 

for a number of disomies. 

In such cases the risk of father<strong>in</strong>g an aneuploid offspr<strong>in</strong>g or 

recurrent abortion is moderately <strong>in</strong>creased. 

Rubes et al, 2002, 2005; Tempest et al, 2009; Uroz et al, 2011 


10







Future 


11 


Constitutional chromosome abnormalities can be 

numerical or structural 

Patients with abnormal karyotypes often present a 

history of repeated spontaneous abortions or 

abnormal sperm parameters 


12

Reciprocal Translocation 

The most frequent structural alterations <strong>in</strong> 

humans and among <strong>in</strong>fertile males 

Carriers produce more unbalanced 

spermatozoa than normal or balanced 

spermatozoa (Moretti et al, 2009) 

55% of the gametes produced by carriers 

will be unbalanced (Bennet et al, 2005) 


13 

Robertsonian Translocation 

 

Carriers show impaired gametogenesis 

to a variable degree 

 

66% of the gametes are expected to be 

unbalanced; average of 15% is observed 

Frydman et al, 2001; Ogur et al, 2006 


14

Inversion 

For pericentric or paracentric <strong>in</strong>version 

carriers the risk of produc<strong>in</strong>g unbalanced 

gametes varies from very low to very 

high depend<strong>in</strong>g on the size of the 

<strong>in</strong>verted chromosome segment 

Anton et al, 2005; Morel et al, 2007; Malan et al, 2006 


15 

Complex Chromosome 

Rearrangements (CCR) 

Structural aberrations <strong>in</strong>volv<strong>in</strong>g three or 

more breakpo<strong>in</strong>ts on two or more 

chromosomes 

Result <strong>in</strong> a high rate of sperm 

chromosome imbalances lead<strong>in</strong>g to 

subfertility 

14.8% of spermatozoa with normal or 

balanced chromosome complement 

Loup et al, 2010 


16

Interchromosomal Effect (ICE) 

Aneuploidies for chromosome pairs not <strong>in</strong>volved <strong>in</strong> the 

chromosomal rearrangement. A potential consequence of 

meiotic disturbances produced by the rearrangements (Lejeune, 

1963, Alfarawati et al, 2012). 

54% of men with Robertsonian translocations, 44% of reciprocal 

translocation carriers and 7% of men with <strong>in</strong>versions have been 

reported to carry an added aneuploidy load <strong>in</strong> the form of an ICE 

(reviewed <strong>in</strong> Anton et al, 2011) 


17 

Numerical Chromosomal Abnormalities 

Kl<strong>in</strong>efelter syndrome (47, XXY) 

The risk of sex chromosome aneuploidy averages 4-6%, much lower 

than what is theoretical expected (Tempest, 2011; Fullerton et al, 

2010, and Blanco et al, 2001) 

47, XYY 

The theoretically expected risk of produc<strong>in</strong>g unbalanced gametes is 

50%, but the real risk measured ranged from 0-4.2% (Benet et al, 

1988; Shi et al, 2001, Blanco et al, 2001) 


18

Males with abnormal karyotype 

Sperm aneuploidy observed <strong>in</strong> these patients is <strong>in</strong>creased, but it is 

lower than what is theoretically expected 

Because: 

• <strong>in</strong> some cases the complex meiotic figures are mechanically 

unresolved and sufficient to block meiotic progression 

• of a selection mechanism aga<strong>in</strong>st aneuploid spermatozoa. 


19 

Conclusion: 

males with abnormal karyotype 

Abnormal karyotypes <strong>in</strong> general are associated with <strong>in</strong>creased risk 

of produc<strong>in</strong>g unbalanced gametes. Hence, a detailed assessment of 

the risk <strong>in</strong>volved <strong>in</strong> each case is of outmost importance 

Counsel<strong>in</strong>g patients about the risk of an abnormal pregnancy 

follow<strong>in</strong>g natural conception or the likelihood of produc<strong>in</strong>g 

embryos suitable for transfer <strong>in</strong> a PGD cycle will help them <strong>in</strong> their 

<strong>in</strong>formed decision mak<strong>in</strong>g 


20







Future 


21 

Infertile males (46, XY) 

After more than a decade of analysis and data collection it is clear 

that <strong>in</strong>fertile men with a normal somatic karyotype have an 

<strong>in</strong>creased risk of sperm aneuploidy 

The risk of produc<strong>in</strong>g chromosomally abnormal sperm <strong>in</strong>creases 

proportionally with the severity of the male <strong>in</strong>fertility 

Sanchez-Castro et al, 2009; Sarrate et al, 2009, Templado et al, 2013 


22

Sperm count 

Strong and <strong>in</strong>verse correlation between 

aneuploidy risk and sperm count 

A 2–6 fold <strong>in</strong>crease for sex chromosome 

disomy and 4x for disomy 21 was reported 

<strong>in</strong> men with severe oligospermia (Sarrate 

et al, 2010; Mougou-Zerelli et al, 2011; 

Durak et al, 2012) 

The risk for aneuploidy is greater <strong>in</strong> the 

testicular sperm of patients with nonobstructive 

azoospermia (Sun et al, 2008) 


23 

Sperm motility 

The relationship between chromosome 

abnormalities and sperm motility is still 

controversial 

No association: Mougou-Zarelli et al, 2011; 

Sarrate et al, 2009 

Moderate risk: Templado et al, 2002; 

Hristova et al, 2002; Bacetti et al, 2005, 

and Collodel et al, 2007 


24

Sperm morphology 

Data regard<strong>in</strong>g association of sperm aneuploidy rate and 

sperm morphology not yet consistent 

Severe sperm morphology abnormalities (macrocephallicmulti-flagellated 

sperm syndrome) display high levels of 

disomy and polyploidy compared to controls 

Perr<strong>in</strong> et al, 2008; Brahem et al, 2011 

Patients with globozoospermia have a moderate to more 

pronounced <strong>in</strong>crease of disomy 

Morel et al, 2004; Brahem et al, 2011 


25 


Semen parameters 

There is a strong and <strong>in</strong>verse correlation between 

aneuploidy risk and sperm count 

The relationship between an <strong>in</strong>creased rate of 

chromosome abnormalities <strong>in</strong> spermatozoa and 

asthenozoospermia or teratozoospermia cannot be 

clearly established 


26

Infertile males (46,XY) 

Increased serum FSH 

Y-microdeletions 

Varicocele 

Cryptorchidism 

Anabolic Abuse 

Chronic Anejaculation 

Increased DFI 

Paternal Age 

Chemotherapy 


27 


Increased serum FSH levels 

Vialard et al (2012) 

Y-microdeletions 

M<strong>in</strong>or et al (2007) and Mateu et al, (2010) 


28


Cryptorhidism 

Moretti et al, 2007 

Varicocele 

Baccetti et al, 2006 


29 

Chemotherapy 

Testicular cancer and Hodgk<strong>in</strong>’s lymphoma are the most common 

malignancies diagnosed dur<strong>in</strong>g reproductive years 

“In general, aneuploidy frequencies decl<strong>in</strong>ed to pretreatment levels 

24 months after treatment <strong>in</strong>itiation” 

Tempest et al, 2008 

“Higher total sperm aneuploidy rate compared with normal men <strong>in</strong> 

testicular tumor patients, even before the onset of the treatment” 

Burello et al, 2011 


30


Anabolic Abuse 

Moretti et al, 2007 

Chronic Anejaculation 

Qui et al, 2012 


31 

Sperm DNA Fragmentation 


32

DNA Fragmentation Index (DFI) 

Several studies reported an association between <strong>in</strong>creased 

sperm aneuploidy and <strong>in</strong>creased DFI 

Muriel et al, 2007; Perr<strong>in</strong> et al, 2008 and 2011; Enciso et al, 2013 

That aneuploidy could trigger DNA fragmentation via an 

apoptotic like process mediated by endogenous nucleases 

Enciso et al, 2013 

Bronnet et al (2012) failed to show any correlation between DFI 

and the aneuploidy rate <strong>in</strong> embryos produced from couples 

with RPL or RIF 


33 

Paternal Age 

It is biologically plausible that chromosomal nondisjunction 

errors should <strong>in</strong>crease with age 

Epidiomiological and the majority of other related studies 

carried out so far failed to determ<strong>in</strong>e the suspected 

negative effect of age on the frequency of disomic sperm 

Buwe et al, 2005; Fonseka et al, 2011; Plastira et al, 2007 


34


<strong>in</strong>fertile males (46,XY) 

In general, the male contribution to the total aneuploidy 

load is considered m<strong>in</strong>or compared to the maternal 

contribution 

However, there are cases where sperm aneuploidy may 

become significant and then the risk of produc<strong>in</strong>g aneuploid 

embryos is <strong>in</strong>creased, while the likelihood of a viable 

pregnancy is considerable decreased 


35 







Future 


36

Occupational/Life style hazards 

Studies of exposures are hampered by: variability of 

dosages, age of donors, heterogeneity between 

<strong>in</strong>dividuals, tim<strong>in</strong>g and length of exposure 



37 

 

 

Smok<strong>in</strong>g/Caffe<strong>in</strong>e/Alcohol: moderate <strong>in</strong>crease 

Robb<strong>in</strong>s et al, 1997 and 2005; Rubes et al, 1998; Shi et al, 2001 

Diazepam, F<strong>in</strong>asteride (Pharmaceuticals): moderate <strong>in</strong>crease 

Collodel et al, 2007; Baumgartner et al, 2001 

 

 

Benzene: moderate <strong>in</strong>crease 

Pesticides <strong>in</strong> general: no <strong>in</strong>crease 

X<strong>in</strong>g et al, 2010 

Harkonen et al, 1999; Smith et al, 2004 

 

Specific Pesticides like Fenvalerate, Carbaryl PCBs and p,p’-DDE: 

moderate <strong>in</strong>crease 

Xia et al, 2004; Xia et al, 2005; McAuliffe et al, 2012 


38


Occupational/Life style hazards 

Only moderate <strong>in</strong>creases <strong>in</strong> the rates of sperm 

aneuploidy have been reported up to date <strong>in</strong> the 

exposed population above the unexposed (1.5– 

3x). 



39 







Future 


40

41 

Mechanisms aga<strong>in</strong>st sperm aneuploidy 

The differences between the paternal and maternal contribution 

to aneuploidy are rather due to more effective checkpo<strong>in</strong>t 

mechanisms <strong>in</strong> spermatogenesis than <strong>in</strong> oogenesis (Uroz et al, 

2012; Templado, 2013) 

It is well known that dur<strong>in</strong>g spermatogenesis various checkpo<strong>in</strong>ts 

are activated to arrest cells with chromosome abnormalities, 

lead<strong>in</strong>g to a reduction <strong>in</strong> gamete production and therefore the 

male <strong>in</strong>fertility (Egozcue et al, 2005) 

Inefficient control mechanisms could therefore be one 

explanation for the <strong>in</strong>creased rate of chromosome abnormalities 

observed <strong>in</strong> some cases (Sarranate et al, 2009) 


42

Is it possible to reduce 

the sperm aneuploidy risk? 

In some cases: YES 

•3 months therapy with FSH 

Piomboni, 2009 

•L-carnit<strong>in</strong>e, acetyl-L carnit<strong>in</strong>e and c<strong>in</strong>noxicam <strong>in</strong> 

OAT patients 

Cavall<strong>in</strong>i et al, 2012 

•Varicocele reversal 

•Folate <strong>in</strong>take 

Baccetti et al, 2006 

Young et al, 2008 


43 

Is it possible to identify 

aneuploid sperms before ICSI? 

In some cases: YES 

•Motile sperm organelle morphology exam<strong>in</strong>ation (MSOME) 

Rita de Cassia et al, 2010; Garolla, 2008; Bartoov et al, 2002 & 2003 

•Hyaluronic Acid b<strong>in</strong>d<strong>in</strong>g sperm selection method for ICSI 

Jakab et al, 2005; Vozdova et al, 2012; Mongkolchaipak et al, 2013 

•Zech-selector chamber 

Seir<strong>in</strong>ger et al,2013 


44

Take home message 

At present, PGD is considered the most valuable option 

for the males at risk <strong>in</strong> order to avoid transmitt<strong>in</strong>g any 

genetic defect to their embryos. 


45 

Male groups worth consider<strong>in</strong>g PGD 

Fertile males who father an aneuploid offspr<strong>in</strong>g 

or recurrent abortions 

Males with structural chromosomal abnormalities 

Males with severe oligospermia or NOA 


46







Future 


47 

Innovative techniques 

Development of more sensitive and <strong>in</strong>formative techniques: 

Further improvement of the <strong>FISH</strong> assay 

New or improved imag<strong>in</strong>g techniques 

Sperm RNA profil<strong>in</strong>g 

Array-based approaches and New Generation Sequenc<strong>in</strong>g 


48

In summary 

• Multicolour <strong>FISH</strong> technique: an effective tool 

• Certa<strong>in</strong> males are at risk 

• New techniques for risk assessment 

• New sperm process<strong>in</strong>g and selection techniques 

• More prospective randomised controlled studies 


49 

Thank you! 

Any questions? 

50

34 

<strong>FISH</strong> on human sperm: technical 

aspects 

Glykeria Samolada

<strong>FISH</strong> on Spermatozoa 

Technical aspects 


Declaration of Interest 

Conflict of <strong>in</strong>terest: None 

2

F I S H: 

Fluorescence In <strong>Situ</strong> <strong>Hybridisation</strong> 

3 

<strong>FISH</strong>: What is it? 

A process, 

which vividly pa<strong>in</strong>ts chromosomes 

or chromosome segments 

with fluorescent molecules 

4

<strong>FISH</strong>: What can it offer? 

Identifies chromosomal abnormalities 

by view<strong>in</strong>g a segment or entire chromosome 

with your own eyes dur<strong>in</strong>g Meta-phase, 

and Inter-phase 

A variety of specimen types can be analysed 

5 

<strong>FISH</strong>: How? 

DNA nucleic acid probe is hybridised 

to a target DNA sequence 

The <strong>in</strong>tact cells are attached 

to a microscope slide 

us<strong>in</strong>g standard cytogenetic methods 

6

<strong>FISH</strong>: How? 

DNA nucleic acid probe is hybridised 

to a target DNA sequence 

The <strong>in</strong>tact cells are attached 

to a microscope slide 

us<strong>in</strong>g standard cytogenetic methods 

7 

What is a Probe? 

a small DNA molecule labeled 

with a marker: 

− which allows identification 

and quantitation 

− that can be hybridised to 

another nucleic acid 

on the basis of base 

complementarity 

8

A DNA probe f<strong>in</strong>ds 

its target <strong>in</strong> place 

Probe 

a ‘molecular tool’ 

9 

Probe Types 

10

<strong>FISH</strong> General Procedure 

1. Preparation of the sample 

2. Denaturation of the target DNA 

3. Denaturation of the probe 

4. <strong>Hybridisation</strong>–Post <strong>Hybridisation</strong> Wash 

5. Fluorescence sta<strong>in</strong><strong>in</strong>g 

6. Exam<strong>in</strong>e slides or store <strong>in</strong> the dark 

11 

Denaturation 

double-stranded DNA is made s<strong>in</strong>gle 

<strong>Hybridisation</strong> 

the formation of a duplex between two 

complementary sequences 

12

<strong>Hybridisation</strong> 

target DNA 

denaturation 

hybridisation 

13 

14

Detection of the signals 

The probe rema<strong>in</strong>s attached to the DNA 

dur<strong>in</strong>g the hybridisation process 

Signals are detected ONLY under a 

fluorescence microscope, equipped with 

the appropriate filter sets 

15 

CCD Camera 

<strong>Fluorescent</strong> Microscope 

Filters 

<strong>FISH</strong> Analysis Software 

16

Sperm-<strong>FISH</strong> 

protocol 

17 

Sperm nuclear architecture 

Unique, well organized nuclear architecture 

different from somatic cells 

Extreme compactness of chromat<strong>in</strong> 

1) each chromosome at dist<strong>in</strong>ct nuclear 

territory 

2) centromeres of non-homologous 

chromosomes 

form chromocentres 

3) telomeres <strong>in</strong>teract with each other form<strong>in</strong>g 

dimers and tetramers 

18

Sperm-<strong>FISH</strong> Procedure 

1. Preparation of the sample 

2. Co-Denature (target DNA & probe) 

3. <strong>Hybridisation</strong> 


5. Exam<strong>in</strong>e slides. Storage <strong>in</strong> the dark 

6. Slide Scor<strong>in</strong>g 

19 

1. Preparation of sample 

Nuclear Decondensation (ND) 

break<strong>in</strong>g disulfide bonds between 

protam<strong>in</strong>es molecules 

optimal mild ND: 1.5-3 fold <strong>in</strong>crease <strong>in</strong> 

nuclear area 

20


Nuclear Decondensation (ND) 

21 


ND protocols 

Neat 0.5M NaOH 10mM DTT-LIS 10mM DTTHep 

22


ND with NaOH 

0.5N NaOH <strong>in</strong>duces mild uniform ND with over 80% of nuclei 

suitable for <strong>FISH</strong> sperm assessment 

Spontaneous ND dur<strong>in</strong>g denaturation and hybridisation should 

be considered <strong>in</strong> <strong>FISH</strong> preparation protocols, as may <strong>in</strong>terfer 

with the proper preparation of the sample 

Comparison of sperm nuclear decondensation protocols us<strong>in</strong>g 

unbiased sampl<strong>in</strong>g and digital image morphometry (2012). S.I. 

Moskovtsev, N. Allad<strong>in</strong>, M. Rogers, J.B.M. Mullen. Mount S<strong>in</strong>ai 

Hosp. Toronto Canada 

23 


Factors Affect<strong>in</strong>g Good Preparation 

1)Sample quality 

2) Fixation technique 

3 parts Methanol + 1 part Acetic Acid 

Always use freshly prepared fixative 

Use with<strong>in</strong> 20 M<strong>in</strong>utes of solution preparation 

3) Optimal Temperature (24°C) 

4) Optimal Relative Humidity (48 to 50%) 

24

2. Co-Denaturation 

(target DNA & probe) 

Factors affect<strong>in</strong>g: 

1) Temperature 

2) Time 

*Formamide & pH (stable 

conditions at co-denaturation) 

25 


Strigency: (tightly bond): the degree to which 

the probe DNA will b<strong>in</strong>d and rema<strong>in</strong> bound 

to the target DNA (after hybridization and at 

Post-Hybridization washes) 

26


Post <strong>Hybridisation</strong> Washes 

freshly daily prepared 

SSC solution temperature: 72±1ºC (not the water 

bath) 

no more that 2 slides at once 

27 


DAPI: A-T rich DNA regions 

Concentration of DAPI/Antifade should not 

“mask” probe's signal 

DAPI fluorescence competes with the signal's 

fluorescence; not brighter 

28

5. Exam<strong>in</strong>e slides 

Microscopy - Bulbs 

• 100 Watt mercury bulb recommended 

• Do not use bulbs over their recommended life 

• Do not use tungsten bulbs 

• Ensure bulb is correctly centred/focused 

• x10 and x60 objectives m<strong>in</strong>imum 

• Phase contrast essential for assess<strong>in</strong>g samples 

• Use s<strong>in</strong>gle filters for clear image / count<strong>in</strong>g 

• Filters last at most 3 - 4 years! 

29 


Scor<strong>in</strong>g criteria 

1. overlapped spermatozoa or sperm heads without a 

well-def<strong>in</strong>ed boundary are not counted 

2. <strong>in</strong> cases of disomy or diploidy, all signals should 

have the same <strong>in</strong>tensity and be separated from each 

other by a distance longer than the diameter of each 

signal 

3. nullisomies are not directly scored and are 

conservatively considered as equivalent to the 

<strong>in</strong>cidence of disomies 

Blanco J. et al (1996) Hum Reprod 11: 722–726 

30


… Scor<strong>in</strong>g criteria 

Furthemore: 

hybridization efficiency greater than 95% 

sperm nuclei of similar size <strong>in</strong>tact 

and not overlapp<strong>in</strong>g 

signals clearly located <strong>in</strong> the nuclei, dist<strong>in</strong>ct, of 

similar size and <strong>in</strong>tensity 

Tempest HG et al, Asian J Androl 2005; 7: 419–25 

31 

(a) <strong>FISH</strong> with chromosome 8 centromere and chromosome 9 centromere specific 

probes. Arrow shows a spermatozoon with 2 chromosomes 9 and 1 chromosome 8. (b) 

<strong>FISH</strong> with chromosome X centromere and chromosome Y centromere specific probes. 

Arrow shows a spermatozoon with 1 chromosome X and 1 chromosome Y. 

Francois Petite at al Journal of Andrology, Vol. 26, No. 2, March/April 2005 

32

<strong>FISH</strong> Advantages 

) Less labor-<strong>in</strong>tensive method 

2) Used at uncultured cells 

3) Own control 

4) No dilution factor 

5) Small sample size 

6) Multiple target analysis 

7) Rather quick (possibly next day result) 

8)Easy to perform 

33 

<strong>FISH</strong> Disadvantages 

Can detect “known” or “suspected” abnormalities 

Not screen<strong>in</strong>g of chromosomal rearrangement 

No structural abnormalities 

No differentiation between nullisomy & hybridisation 

failures 

Only 1-3 probe-signal / sperm head 

Cost 

Needs experienced technician 

Scor<strong>in</strong>g – Time consum<strong>in</strong>g 

(low aneuploidy rate/chromosome affected by sample's 

size) 

Automated Sperm-<strong>FISH</strong>? 

34

<strong>FISH</strong> 

Results Interpretation 

Sperm <strong>FISH</strong> def<strong>in</strong>es the proportion of aneuploidy present 

<strong>in</strong> the ejaculates of males with other (genetic/cl<strong>in</strong>ical) 

problems 

The goal: to identify “errors” and the “at-risk” males 

Quantitative vs Qualitative analyses 

“...<strong>FISH</strong> analysis of spermatozoa should be used as a tool of genetic screen<strong>in</strong>g <strong>in</strong> 

<strong>in</strong>fertile patients. Significant differences <strong>in</strong> the rates of chromosome abnormalities 

with respect to controls should be taken <strong>in</strong>to consideration regardless of the 

numerical value. When abnormal results are obta<strong>in</strong>ed, <strong>in</strong>dividuals should be 

identified as ‘‘at risk’’ and the couple should be advised about the available 

techniques of preimplantation and prenatal genetic diagnosis” 

Sarrate et al, 2010 

35 

36

Figure 5. Model of chromosome organization <strong>in</strong> human spermatozoa. 

Compact CTs (filled contours) have overall hairp<strong>in</strong> conformations (chromosome paths <strong>in</strong>dicated by dashed l<strong>in</strong>es) with the p and q telomere/subtelomere 

doma<strong>in</strong>s (orange circles) form<strong>in</strong>g dimers at nuclear periphery. Gene-rich CHRs – rosy, gene-poor – 37 <strong>in</strong>digo. CTs are connected via 

centromeres/peri-centromeres (green circles and l<strong>in</strong>es) <strong>in</strong>to arrays and have a fixed l<strong>in</strong>ear order which determ<strong>in</strong>es the longitud<strong>in</strong>al position<strong>in</strong>g of 

chromosomes. 

Mudrak OS, B. Nazarov I, Jones EL, Zalensky AO (2012) Position<strong>in</strong>g of Chromosomes <strong>in</strong> Human Spermatozoa Is Determ<strong>in</strong>ed by Ordered 

Centromere Arrangement. PLoS ONE 7(12): e52944. doi:10.1371/journal.pone.0052944 

http://www.plosone.org/article/<strong>in</strong>fo:doi/10.1371/journal.pone.0052944 

38

Happy Fish<strong>in</strong>g 

39 

Thank you! 

40 

Any questions?

55 

<strong>FISH</strong> technique: 

Standard Operat<strong>in</strong>g procedure, 

materials and methods, report<strong>in</strong>g, 

Internal and External Quality 

Control for <strong>FISH</strong> 

Glykeria Samolada

<strong>FISH</strong> on Spermatozoa 

SOP 

Preparation 

6. Procedure 

6.1 Preparation 

Wash your sample, dilut<strong>in</strong>g 1ml with 4ml PBS and centrifuge for 7m<strong>in</strong> at 2000 rpm. 

Remove the supernatant and resuspend the pellet. Dilute it with 1ml fresh PBS and centrifuge 

for 7m<strong>in</strong> at 2000 rpm. 

Repeat the second wash for one more time. 

TIP: Resuspend your pellet before add<strong>in</strong>g the fresh PBS. 

Dur<strong>in</strong>g the last centrification, prepare your fixative solution. Fixative should be cool and 

fresh. 

FIXATIVE SOLUTION 

Prepare FRESH fixation solution 3:1 Methanol/Acetic Acid 

For f<strong>in</strong>al volume of 4 ml mix 

• 3ml of methanol 

• 1ml of CH 3COOH 

At the end of the 3 rd wash remove all the supernatant and resuspend the pellet. 

Add your fixative drop-to-drop, to dilute your sample. 

Leave the sample on the fridge (4 o C) for 1 hour. 

After 1 hour, remove the sample from the fridge and centrifuge for 3m<strong>in</strong>. 

Remove all the supernatant and resuspend the pellet. 

Add fresh fixative drop-to-drop. 

Clean your slides at Ethanol and pour your sample drop-to-drop to have a good spread<strong>in</strong>g. 

Hands-on Workshops, Thessaloniki, 30.08 – 5.09 2014 2

Tip for spread<strong>in</strong>g 

• Keep your slide humid 

• Keep your sample quite high and leave it to fall on the slide 

Observe the slides carefully under the microscope to decide how much time each slide it will 

need for treatment. 

NaOH helps to remove any debris from your sample and also de-condense the spermatozoa. 

Samples with condensed spermatozoa heads or a lot of debris need to be treated with NaOH 

for a longer period of time (2-8 m<strong>in</strong>). 

TIP: Cryopreserved samples are usually more compact than fresh samples, so they need a 

longer treatment with NaOH at this stage. 

Hands-on Workshops, Thessaloniki, 30.08 – 5.09 2014 3 

Treatment 

At this stage, samples are treated with NaOH 0.5M. Each slide is treated for the period of 

time that was decided previously (under the microscope). 

After treatment with NaOH, samples are washed twice <strong>in</strong> fresh SSC 2X (SSC consists of NaCl 

and Sodium Citrate), <strong>in</strong> order to remove the rema<strong>in</strong><strong>in</strong>g NaOH from your samples. Each wash 

lasts for 5 m<strong>in</strong>utes 

F<strong>in</strong>ally, samples are dried <strong>in</strong> ethanol (gradually 70%, 90% and 100%) for 5 m<strong>in</strong>utes <strong>in</strong> each 

dilution. 

Observe the slides carefully under the microscope and decide where the probe is go<strong>in</strong>g to be 

added. Mark the selected region, at which your sample is better treated 


Co-denaturation 

Firstly, add 3- 10µl of your probe <strong>in</strong> each marked region and cover with glass coverlsip (small 

round or 20X20). Avoid expos<strong>in</strong>g your samples on light, to keep the levels of fluorescence for 

sample’s count<strong>in</strong>g. 

Before the hybridization, samples should be co-denaturated to obta<strong>in</strong> s<strong>in</strong>lge-stranded DNA 

both at probes and at the sperm DNA. For co-denaturation, slides are placed <strong>in</strong> a hot plate 

at 74 o C. (For chromosome 18 leave the slides at 74 o C for 2 m<strong>in</strong>utes and for the chromosomes 

X, Y, 13 and 21 for 5 m<strong>in</strong>utes, or accord<strong>in</strong>g to probe’s supplier guides) . 


Hybridization 

For the hybridisation, <strong>in</strong>cubate the samples overnight <strong>in</strong>37 o C, 

<strong>in</strong>side a humid and dark box. 


Post Hybridization Wash 

The aim of this stage is to remove all the non-specific hybridized sites that 

might have been created dur<strong>in</strong>g the hybridization. 

Remember to keep your samples <strong>in</strong> dark dur<strong>in</strong>g the whole procedure, as light 

will reduce the fluorescence of the probes 

Firstly, place your samples <strong>in</strong> 2X SSC for some seconds, just to remove the 

coverslips easily. 

Adjust the temperature of SSC solution <strong>in</strong> the jar. 

Put your slides <strong>in</strong> jars with SSC (for chromosome 18 wash <strong>in</strong> 0.25M SSC and for 

chromosomes 13, 21, X and Y <strong>in</strong> 0.4M SSC). The jars should be <strong>in</strong>cubated <strong>in</strong> 

water-bath at 74 o C for 2 m<strong>in</strong>utes exactly. 

After the bath, wash with Water For Injection or Phosphate Buffered for 1 

m<strong>in</strong>ute, <strong>in</strong> order to remove the rema<strong>in</strong><strong>in</strong>g SSC without agitation. 

F<strong>in</strong>ally, samples are dried <strong>in</strong> ethanol (gradually 70%, 90% and 100%) for 2 

m<strong>in</strong>utes <strong>in</strong> each dilution. 


Sta<strong>in</strong><strong>in</strong>g 

At this stage, spermatozoa are sta<strong>in</strong>ed, to be visualized under the microscope. 

For sta<strong>in</strong><strong>in</strong>g, 3-10μl of DAPI Antifade are added <strong>in</strong> each marked region and 

covered with glass coverslip. 

It is important to discrete the marg<strong>in</strong>s of each cell, so as to be sure of the 

sign that you get. 

Coverslips are placed carefully above each marked region and are secured <strong>in</strong> 

place with a little polish. 


Scor<strong>in</strong>g - Count<strong>in</strong>g 

Fields should be found at 10X or 20X magnification. Then, magnification should 

be <strong>in</strong>creased to 40X and gradually to 100X with oil, to count the spots. 

There should be only one spot of each chromosome <strong>in</strong> every sperm cell. 

(Chromosomes 18, X and 13at our experiment emit green colour, whereas 

chromosomes Y and 21 emit red colour). 

TIP: 

•Spot count<strong>in</strong>g should be careful. The marg<strong>in</strong>s of the cells should be clear <strong>in</strong> 

order to know that the spots come from chromosomes. 

•2 spots <strong>in</strong> a cell should be clearly separated and have the same <strong>in</strong>tensity <strong>in</strong> 

order to be considered as aneuploid. (Blanco et al, 1996) 


Sperm <strong>FISH</strong> 

QC 

Use always your control 



External QC 



External QC 


Happy Fish<strong>in</strong>g 


Thank you! 

14 

Any questions?

Issue Date: 

05/12/2012 

Version: 

1 

Rev.: 

0 

SOP-LAB-01: <strong>FISH</strong> ON HUMAN SPERM 

Page #: 

1 out of 7 

Prepared by: Glykeria Samolada Edited by: Vassilis Katsares Approved by: Alexia Chatziparasidou 

SOP: SOP-LAB-02 

<strong>FISH</strong> ON HUMAN SPERM 

Version: 01 

Revision: 00 

Issue Date: 05-12-2012 

Date of last revision: 

Prepared by: 

Edited by: 

Approved by: 


Vassilis Katsares 


SOP-LAB-02: <strong>FISH</strong> ON HUMAN SPERM/01/00/05-12-2012

Issue Date: 

05/12/2012 


1 

Rev.: 

0 


Page #: 

2 out of 7 


Revision Table 

Date Version Description of 

changes 

05-12-2012 1 - 

Name and Signature 

for the changes 

Name and signature for 

the approval 


Issue Date: 

05/12/2012 


1 

Rev.: 

0 


Page #: 

3 out of 7 


Table of Contents 

Paragraph 

Page 

Purpose 4 

Scope/Field of Application 4 

Def<strong>in</strong>ition and Acronyms 4 

Responsibilities 4 

Materials Required 4 

Procedure 5 

Documentation 7 

Reference Procedures 8 

References 8 

Revision History 8 


Issue Date: 

05/12/2012 


1 

Rev.: 

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1. Purpose 

The purpose of this procedure is to describe the technique of <strong>FISH</strong> on human spermatozoa. 

2. Scope/Field of Application 

This procedure is taken place <strong>in</strong> the IVF lab and the genetic material to be exam<strong>in</strong>ed is isolated from human 

spermatozoa. 

3. Def<strong>in</strong>itions and Acronyms 

IVF: In Vitro Fertilization 

PBS: Phosphate Buffer Sal<strong>in</strong>e 

SSC: Sodium chloride – Sodium Citrate 

4. Responsibilities 

The current procedure is performed only by properly tra<strong>in</strong>ed personnel (geneticists and technicians), who are well 

experienced <strong>in</strong> sperm <strong>FISH</strong>. The IVF unit that provides this service, as well as the collaborated laboratories for the 

genetic analysis, must be certified accord<strong>in</strong>g to ISO 9001 and should be able to provide patients with genetic counsel<strong>in</strong>g 

and all necessary consent forms with clear <strong>in</strong>formation on all possible outcomes and results. 

5. Materials Required 

Ma<strong>in</strong>tenance-consumables-consent forms: 

Prior to this procedure all equipment to be used should be validated and ma<strong>in</strong>ta<strong>in</strong>ed accord<strong>in</strong>g to the manufacturer’s 

<strong>in</strong>structions. All consumables, plastic ware and media used, should be CE marked and <strong>in</strong>dividually packaged or 

aliquoted, when possible. All consent forms concern<strong>in</strong>g the patients counsel<strong>in</strong>g, the procedure, the diagnosis and the 

possible outcomes should be clearly stated and signed by the patient(s). 

Hardware: 

Inverted microscope, <strong>Fluorescent</strong> microscope with filters, light source, hot plate, centrifuge. 

Sperm /cell manipulation 

2-20 µl micropipettes, 20-200 µl micropipettes, filtered tips (20 µl and 200 µl), 

Miscellaneous: 

Laboratory footwear and outfit, masks (Hospital and Home care Surgical Face masks), s<strong>in</strong>gle use robes, laboratory caps, 

sterile surgical gloves (latex, powder free). 


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6. Procedure 

6.1 Preparation 

Wash your sample, dilut<strong>in</strong>g 1ml with 4ml PBS and centrifuge for 7m<strong>in</strong> at 2000 rpm. 

Remove the supernatant and resuspend the pellet. Dilute it with 1ml fresh PBS and centrifuge for 7m<strong>in</strong> at 2000 rpm. 

Repeat the second wash for one more time. 

TIP: Resuspend your pellet before add<strong>in</strong>g the fresh PBS. 

Dur<strong>in</strong>g the last centrification, prepare your fixative solution. Fixative should be cool and fresh. 

FIXATIVE SOLUTION 

Prepare FRESH fixation solution 3:1 Methanol/Acetic Acid 

For f<strong>in</strong>al volume of 4 ml mix 

• 3ml of methanol 

• 1ml of CH3COOH 

At the end of the 3 rd wash remove all the supernatant and resuspend the pellet. 

Add your fixative drop-to-drop, to dilute your sample. 

Leave the sample <strong>in</strong> the fridge (4 o C) for 1 hour. 

After 1 hour, remove the sample from the fridge and centrifuge for 3m<strong>in</strong> at 2000 rpm. 

Remove all the supernatant and resuspend the pellet. 

Add fresh fixative drop-to-drop. 

Clean your slides with Ethanol and pour your sample drop-to-drop to have a good spread<strong>in</strong>g. 

TIPS FOR SPREADING: 

• Keep your slide humid 

• Keep your sample quite high and leave it to fall on the slide 

Observe the slides carefully under the microscope to decide how much time each slide it will need for treatment. 

NaOH (0.5M) helps to remove any debris from your sample and also de-condense the spermatozoa. Samples with 

condensed spermatozoa heads or a lot of debris need to be treated with NaOH for a longer period of time (2-8 m<strong>in</strong>). 

TIP: Cryopreserved samples are usually more compact than fresh samples, so they need a longer treatment with NaOH 

at this stage. 

6.2 Treatment 

At this stage, samples are treated with NaOH 0.5M. Each slide is treated for the period of time that was decided 

previously (under the microscope). 

After treatment with NaOH, samples are washed twice <strong>in</strong> fresh SSC 2X (SSC consists of NaCl and Sodium Citrate), <strong>in</strong> 

order to remove the rema<strong>in</strong><strong>in</strong>g NaOH from your samples. Each wash lasts for 5 m<strong>in</strong>utes. 

F<strong>in</strong>ally, samples are dried <strong>in</strong> ethanol (gradually 70%, 90% and 100%) for 5 m<strong>in</strong>utes <strong>in</strong> each dilution. 

Observe the slides carefully under the microscope and decide where the probe is go<strong>in</strong>g to be added. Mark the selected 

region, at which your sample is better treated. 


Issue Date: 

05/12/2012 


1 

Rev.: 

0 


Page #: 

6 out of 7 


6.3 Co-denaduration 

Firstly, add 3-10µl of your probe <strong>in</strong> each marked region and cover with glass coverlsip (small round or 20X20). Avoid 

expos<strong>in</strong>g your samples on light, to keep the levels of fluorescence for sample’s count<strong>in</strong>g. 

Before the hybridisation, samples should be co-denaturated to obta<strong>in</strong> s<strong>in</strong>gle-stranded DNA both at probes and at the 

sperm DNA. For co-denaturation, slides are placed on a hot plate at 74 o C. (For chromosome 18 leave the slides at 74 o C 

for 2 m<strong>in</strong>utes and for the chromosomes X, Y, 13 and 21 for 5 m<strong>in</strong>utes, or accord<strong>in</strong>g to probe’s supplier guides). 

6.4 <strong>Hybridisation</strong> 

For the hybridization, <strong>in</strong>cubate the samples overnight <strong>in</strong> 37 o C, <strong>in</strong>side a humid and dark box. 

6.5 Post-<strong>Hybridisation</strong> Wash 

The aim of this stage is to remove all the non-specific hybridized sites that might have been created dur<strong>in</strong>g the 

hybridization. 

Remember to keep your samples <strong>in</strong> dark dur<strong>in</strong>g the whole procedure, as light will reduce the fluorescence of the probes 

Firstly, place your samples <strong>in</strong> 2X SSC for some seconds, just to remove the coverslips easily. 

Adjust the temperature of SSC solution <strong>in</strong> the jar. 

For the post-hybridisation wash, put your slides <strong>in</strong> jars with SSC (for chromosome 18 wash <strong>in</strong> 0.25M SSC and for 

chromosomes 13, 21, X and Y <strong>in</strong> 0.4M SSC). The jars should be <strong>in</strong>cubated <strong>in</strong> water-bath at 74 o C for 2 m<strong>in</strong>utes exactly. 

After the bath, wash with Water For Injection or Phosphate Buffered for 1 m<strong>in</strong>ute, <strong>in</strong> order to remove the rema<strong>in</strong><strong>in</strong>g SSC 

without agitation. 

F<strong>in</strong>ally, samples are dried <strong>in</strong> ethanol (gradually 70%, 90% and 100%) for 2 m<strong>in</strong>utes <strong>in</strong> each dilution. 

6.6 Sta<strong>in</strong><strong>in</strong>g 

At this stage, spermatozoa are sta<strong>in</strong>ed, to be visualized under the microscope. 

For sta<strong>in</strong><strong>in</strong>g, 3-10µl of DAPI Antifade are added <strong>in</strong> each marked region and covered with glass coverslip. 

It is important to discrete the marg<strong>in</strong>s of each cell, so as to be sure of the sign that you get. 

Coverslips are placed carefully above each marked region and are secured <strong>in</strong> place with a little polish. 

6.7 Scor<strong>in</strong>g & Count<strong>in</strong>g 

Fields should be found at 10X or 20X magnification. Then, magnification should be <strong>in</strong>creased to 40X and gradually to 

100X with oil, to count the spots. 

There should be only one spot of each chromosome <strong>in</strong> every sperm cell. (Chromosomes 18, X and 13at our experiment 

emit green colour, whereas chromosomes Y and 21 emit red colour). 

TIP: 

• Spot count<strong>in</strong>g should be careful. The marg<strong>in</strong>s of the cells should be clear <strong>in</strong> order to know that the spots come 

from chromosomes. 

• 2 spots <strong>in</strong> a cell should be clearly separated and have the same <strong>in</strong>tensity <strong>in</strong> order to be considered as aneuploid. 

(Blanco et al, 1996) 


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7. Documentation 

Nonconform<strong>in</strong>g samples are recorded on accompany<strong>in</strong>g submission forms. Equipment problems are recorded <strong>in</strong> the 

appropriate equipment ma<strong>in</strong>tenance log. All nonconformances are recorded <strong>in</strong> the Corrective Action Request form. 

Root cause analysis and closed loop corrective actions taken to prevent recurrence of nonconformances are recorded as 

described <strong>in</strong> the proper SOP. 

Required Record 

Nonconformance Report 

“Calibration Void – Do Not Use” label 

“Out of Service – Do Not Use” label 

Custodian 

Responsible Manager 



8. Reference Procedures 

9. References 

Holmes, J.M. and Mart<strong>in</strong>, R.H. (1993) Aneuploidy detection <strong>in</strong> human sperm nuclei us<strong>in</strong>g fluorescence <strong>in</strong> situ 

hybridization. Hum Genet 91:20-24. 

Egozcue, J., Blanco, J. and Vidal, F. (1997) Chromosome studies <strong>in</strong> human sperm nuclei us<strong>in</strong>g fluorescence <strong>in</strong> situ 

hybridization (<strong>FISH</strong>). Hum Reprod Update 3: 441-452. 

Blanco, J., Egozcue, J. and Vidal, F. (1996) Incidence of chromosome 21 disomy <strong>in</strong> human spermatozoa as determ<strong>in</strong>ed 

by fluorescent <strong>in</strong> situ hybridization. Hum Reprod 11: 722-726. 

Mart<strong>in</strong>, R.H. (2007) The cl<strong>in</strong>ical relevance of sperm aneuploidy, In: Carrell, D. (ed.), The Genetics of Male Infertility, 

Humana Press: Totowa, NJ, pp. 127-144. 

Munné, S. (2003) Preimplantation Genetic Diagnosis and Human Implantation—A Review Placenta 24: S70-S76. 

10. Revision History 

Revision 0 



Hands-on workshops <strong>in</strong> specialised ART laboratory techniques 

August 31 - September 5, 2014 Thessaloniki, Greece 

70 

# Name Title Institute e-mail Country 

1 Ms. Elisabeth Foluke Philip Embryologist Georges Memorial Medical Centre folu25@yahoo.com Nigeria 

2 Ms. Nicole Klauke Embryologist K<strong>in</strong>derwunschzentrum an der Gedächtniskirche / Berl<strong>in</strong> nicole-klauke@freenet.de Germany 

3 Ms. Adedamilola Adesewa Atiba Embryologist NORDICA FERTILITY CENTER, LAGOS adedamilola.atiba@nordicalagos.org Nigeria 

4 Ms. Styliani Sgoura Lab technician Embryolab s.sgoura@embryolab.eu Greece 

5 Ms. Aimilia Vorniotaki Lab technician Embryolab a.vorniotaki@embryolab.eu Greece

Our generous Sponsors 

71

72 

General <strong>in</strong>formation 

Target Audience 

Embryolab Academy Hands-on Workshops are designed for reproductive scientists, 

embryologists, lab technicians, geneticists and cl<strong>in</strong>icians who desire to improve their knowledge 

and practical skills <strong>in</strong> specialised laboratory techniques used <strong>in</strong> a human ART sett<strong>in</strong>g. 

Prom<strong>in</strong>ent teachers 

Embryolab Academy strives to provide evidence based scientific knowledge presented by an 

<strong>in</strong>ternational panel of prom<strong>in</strong>ent experts. Our teachers have been carefully selected on the basis 

of the importance and weight of their scientific work <strong>in</strong> the specific doma<strong>in</strong>s of <strong>in</strong>terest as well 

as their excellent educational skills and their day to day hands-on experience <strong>in</strong> different 

laboratory techniques <strong>in</strong> IVF. 

Venue 

The Hands-on Workshops will be held at Embryolab Academy, 173-175 Ethnikis Antistaseos, 551 34 

Kalamaria, Thessaloniki, Greece. 

Transport to Embryolab Academy dur<strong>in</strong>g your Workshop 

Transport from the City hotel to and from the Workshop Venue is <strong>in</strong>cluded <strong>in</strong> the registration 

fee. Embryolab Academy will confirm morn<strong>in</strong>g time and place of departure at your hotel. 

Thessaloniki 

Thessaloniki is the second-largest city <strong>in</strong> Greece and the capital of the region of Central 

Macedonia. Greece’s second major economic, <strong>in</strong>dustrial, commercial and political centre and a 

major transportation hub for the rest of southeastern Europe. The city is renowned for its 

festivals, events and vibrant cultural life <strong>in</strong> general, and is considered to be Greece’s cultural 

capital. 

Climate 

Day temperatures <strong>in</strong> September range from 15°C to 30°C, overall days are sunny and dry. 

Workshop language 

The official language of the Workshop is English. 

Liability 

The Workshop secretariat and organiser cannot assume liability for personal accidents, loss of 

or damage to private property of participants, either dur<strong>in</strong>g, or directly aris<strong>in</strong>g from the 

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because of air strikes or serious adverse events. 

Time zone 

The time zone <strong>in</strong> Greece is Eastern Europe Time Zone: UTC/GMT +2 hours.

73 

Certificate of attendance 

A Certificate of attendance will be awarded to the delegates after attend<strong>in</strong>g the full workshop 

programme. After complet<strong>in</strong>g the evaluation form of the workshop, your certificates will be 

awarded to you. 

The Hands-on Workshops are runn<strong>in</strong>g under the auspices, ASEBIR, the Spanish Society for the 

Study of Biology of Reproduction 

Workshop Secretariat 



<strong>in</strong>fo@embryolab-academy.org 

www.embryolab-academy.org 

Workshop Venue 

Embryolab Academy at Embryolab – Campus, Thessaloniki, Greece 



www.embryolab-academy.org

74 

About Embryolab Academy 

Embryolab Academy is a non-profit foundation, focused on education, tra<strong>in</strong><strong>in</strong>g and research <strong>in</strong> 

assisted reproduction. Embryolab Academy promotes close collaboration between 

embryologists, andrologists, fertility specialists, scientists and human geneticists to improve 

quality, safety and accuracy <strong>in</strong> assisted reproduction techniques, and reproductive medic<strong>in</strong>e. 

The objectives of the Embryolab Academy are to: 

Organise and host International Workshops, focused on state of the art assisted 

reproduction techniques, and reproductive medic<strong>in</strong>e. 

Provide <strong>in</strong>dividual, hands-on courses and tra<strong>in</strong><strong>in</strong>g <strong>in</strong> assisted reproduction techniques, 

and reproductive medic<strong>in</strong>e. 

Promote and coord<strong>in</strong>ate research <strong>in</strong> the field of assisted reproduction techniques, and 

reproductive medic<strong>in</strong>e. 

Promote communication, collaboration and exchange of expertise between different 

specialist, work<strong>in</strong>g <strong>in</strong> assisted reproduction techniques, and reproductive medic<strong>in</strong>e. 

Embryolab Academy MKO 



www.embryolab-academy.org

WORKSHOP BOOK Fluorescent in Situ Hybridisation - FISH - on Human Sperm

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