BLASTOCOELE: THE EMBRYONIC FLUID COMPARTMENT. A

24.02.2015
LEARNING OBJECTIVES
1) UNDERSTAND THE METHOD TO COLLECT
BLASTOCOELE FLUID
2) HOW TO DETECT GENOMIC DNA IN BLASTOCOELE
FLUID
3) WHAT ARE THE APPLICATION OF DNA IN
BLASTOCOELE
Genomic DNA in human blastocele fluid
Dott. Simone Palini
Senior clinical embryologist
O.U. Physiopathology of Reproduction
Cervesi Hospital-Cattolica AUSL Romagna (RN) ITALY
[email protected]
THE IDEA
THE IDEA
A negative correlation between
blastocelic volume and outcome
measures has been attributed to an
increased likelihood of intracellular ice
formation in an inadequately
dehydrated blastocoele. Consequently, a
process called assisted shrinkage was
developed to reduce blastocelic volume
prior to vitrification.
Kader et al. 2009
THE IDEA
THE IDEA
Chen et al. 2005
0.3/1.5 nl BF
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24.02.2015
QUESTIONS ABOUT
1. There is genomic DNA in human Blastocoele fluid
(BF)?
1. There is genomic DNA in human Blastocoele fluid (BF)?
2. How much DNA?
A. PCR real time single copy gene GAPDH
A. PCR real time multi copy gene: TBC1D3
(chromosome 17) and TSPY1 (chromosome Y)
3. PGS on BF?
4. PGD on BF? DNA in BF represent embryo’s DNA?
A. WGA, PCR and microarray CGH
5. New idea about non invasive diagnosis?
1. There is genomic DNA in human Blastocoele fluid
(BF)?
A. PCR real time single copy gene GAPDH
1. There is genomic DNA in human Blastocoele fluid
(BF)?
A. PCR real time single copy gene GAPDH
1 maggio 2012
Gene target
Primer name
5’---3’
Amplicon
length (bp)
GAPDH
NG_007073
GAPDH-F
CCATGTTCGTCATGGGTGTG
183
GAPDH-R
GGTGCTAAGCAGTTGGTGGTG
These resulte indicate that 9/16 (56%) blastocoele
fluid samples contained PCR-amplifiable DNA and that,
in at least three samples, this DNA was fragmented
Palini et al 2013
1. There is genomic DNA in human Blastocoele fluid
(BF)?
B. PCR real time multi copy gene TBC1D3
(chromosome 17) and TSPY1 (chromosome Y)
Gene target
Primer name
5’---3’
Amplicon
length (bp)
TSPY1
NG_027958
TSPY1_F
TGTAAGTGACCGATGGGCAG
60
TSPY1-R
AACTCCCCTTTGTTCCCCAA
TBC1D3
NW003315949
TBC1D3_F
GGGCAAGAGGTCATCTGAGC
TBC1D3_R
TGCTTCCTTAATGTGCCGCT
below 183 bp
Palini et al 2013
1. There is genomic DNA in human Blastocoele fluid
(BF)?
B. PCR real time multi copy gene TBC1D3
(chromosome 17) and TSPY1 (chromosome Y)
1.
Among the 26/31
samples that showed
the TBC1D3 product,
17 samples also
showed amplification
of TSPY1
2.
65.4% males and
34.6% females
66
Palini et al 2013
Palini et al 2013
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24.02.2015
1. There is genomic DNA in human Blastocoele fluid
(BF)?
1. There is genomic DNA in human Blastocoele fluid
(BF)?
C. WGA, PCR and Microarray CGH
ANSWER TO THE FIRST QUESTION
BF contains by PCR amplifiable DNA and could
be used to sex determinations avoiding X-linked
diseases, after the necessary validation!!
To investigate whether WGA products were a suitable template for subsequent comprehensive
chromosome analysis, amplified samples from FOS2 and VAN5 were labelled and tested using a
well-established microarray CGH approach. Chromosome screening was accomplished in both
cases, producing data that suggested that both embryos were male (i.e. a normal number of
copies of the X and Y chromosomes detected). These results were concordant with data from
the same two WGA samples obtained using PCR amplification of TSPY1 on the Y chromosome.
Additionally, micro- array CGH results indicated that both DNA samples were aneuploid. The
predicted karyotype was 47,XY,+22 (male with trisomy 22) in one case and 46,XY,-1,-10,+11,+16
(male with complex aneuploidy) in the other
Palini et al 2013
2. How much DNA?
2. How much DNA?
The amount of DNA in the blastocoele fluid was estimated
with RotorGene 6000 software, where the positive control for
each run was the reference for calibration curve adjustments.
Based on the TBC1D3 calibration curve, this study roughly
estimated that 0.8–55 pg (median 7.3 pg) of genomic DNA
were present in the blastocoele fluid samples. A similar range
(3–85 pg; median 12.2 pg) was obtained with the TSPY1
calibration curve. Based on an unpaired t-test with the Welch
correction, the mean values from the two calibration curves
did not differ significantly.
ANSWER TO THE SECOND QUESTION
9.9 pg of genomic DNA per sample
The combined results showed a total median value of
genomic DNA per sample.
Palini et al 2013
Palini et al 2013
3. PGS on BF?
3. PGS on BF?
N°
SAMPLE
KARYOTYPE
1
BF
46 XY
TE
46 XY -22
BF
46 XXY +14
TE
46 XY
BF
46 XY
TE
46 XY
BF
45 X0
TE
46 XY
2
3
4
BF
Non invasive approch
TE
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3. PGS on BF?
OUR HYPOTHESES
- DNA is fragmented
- DNA could come from apoptotic cells
- Is not the right way or the right template to
study DNA from BF
- DNA in BF is the results of TE and ICM
3. PGS on BF?
After whole-genome amplification (WGA), DNA was
detected in 39 BFs out of 51 (76.5%), providing 39
complete sets with the chromosome status available for
PBs or blastomeres, BFs, and TE cells. The mean amount
of DNA detected, amplified from the BF, was 900.38
ng/mL (range 876.3– 939.5 ng/mL).
array CGH (24sure; Bluegnome).
Gianaroli L. et al, Fertility and Sterility® Vol. 102, No. 6, December 2014 0015-0282/$36.00
3. PGS on BF?
3. PGS on BF?
3. PGS on BF?
3. PGS on BF?
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24.02.2015
3. PGS on BF?
4. PGD on BF? DNA in BF represent embryo’s DNA?
ANSWER TO THE THIRD QUESTION
1.
all ours results show 67% BF’s chromosomal set
correspond to TE…more data are needed and the
technique will be optimized
2 probably BF will not be used to do PGS with our
knowledge/instruments or approch
A. PGD on human embryos: sex determination
by PCR using BF sex confirmed with scan and
CGH microarray
B. PGD on equine embryos: sex determination
by PCR using BF
C. PGD on human embryos: point mutation
detection (MTHFR)
3 looking for the right approach, take care, don’t discarts
embryos that could be viable
4. PGD on BF? DNA in BF represent embryo’s DNA?
A. PGD on human embryos: sex determination by PCR using
BF, sex confirmed with scan and CGH microarray
1.
Among the 26/31
samples that showed the
TBC1D3 product, 17/26
samples also showed
TSPY1 amplification
2.
65.4% males and 34.6%
females
1.
to date 5/5 of warming
embryos that gave
pregnancy confirmed the
sex diagnosis obtain
throught BF analisy
4. PGD on BF? DNA in BF represent embryo’s DNA?
B. PGD on equine embryos: sex determination
by PCR using BF
4. PGD on BF? DNA in BF represent embryo’s DNA?
A. PGD on human embryos: sex determination by PCR using
BF, sex confirmed with scan and CGH microarray
4. PGD on BF? DNA in BF represent embryo’s DNA?
B. PGD on equine embryos: sex determination
by PCR using BF
The ge- netic sex was determined in 11 of the 13 (84.6%) and four of the five (80%) blastocele fluid
samples obtained from in vivo– and in vitro–produced embryos, respectively.
Eight of the eleven blastocele fluid samples from the in vivo–produced embryos that amplified were
diagnosed as males and three were diagnosed as females, whereas two out of the four amplified
samples from the in vitro– produced embryos were diagnosed as males and two as females.
C. Herrera et al. / Theriogenology 83 (2015) 415–420
C. Herrera et al. / Theriogenology 83 (2015) 415–420
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4. PGD on BF? DNA in BF represent embryo’s DNA?
B. PGD on equine embryos: sex determination
by PCR using BF
Seven of the 13 in vivo–produced embryos were cultured in vitro
and observed every 24 hours for post– blastocele fluid aspiration
survival. Six of seven embryos reexpanded at 24 hours of in vitro
culture and increased their diameter at 48 and 72 hours. The
average per- centage increase in diameter at the end of the
culture period was 73.7%, from a mean of 633 mm to a mean of
1100 mm. Four of the six reexpanded embryos hatched in vitro
during the culture period. Figure 3 shows one of the in vivo–
produced embryos hatching, after 48 hours of in vitro culture.
C. Herrera et al. / Theriogenology 83 (2015) 415–420
4. PGD on BF? DNA in BF represent embryo’s DNA?
C. PGD on human embryos: point mutation (MTHFR)
WGA picoplex
Embryo ID
Sample amplified by WGA
MTHFR amplification
C677T genotype
1
Blastocoele fluid
+
C/T
2
Blastocoele fluid
+
C/T
Blastocoele fluid
+
C/T
Biopsy
+
C/T
Blastocoele fluid
--
n.a.
Blastocoele fluid
+
C/T
Biopsy
+
C/T
3
Blastocoele fluid
+
C/T
5
Blastocoele fluid
+
T
6
Blastocoele fluid
--
n.a.
7
Blastocoele fluid
--
n.a.
WGA 1° method
1IB
2IB
3IB
WGA picoplex
1
225
3
4
526
6
727
8
9
1028
11
1229
13
14
30
15
16
1731
18
1932
20
21
2233
23
2434
25
26
2735
28
2936
30
31
37
32
33
3438
35
3639
37
38
3940
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Introduction
The preimplantation genetic diagnosis (PGD) of a multitude of hereditary genetic disorders,
currently relies on invasive procedures such as blastomeres or trophectoderm biopsy, which are
associated with a small but significant risk of embryo’s damage (Labonte, 2012;Kirkegaard et al.,
4. PGD
DNA
BF represent
embryo’s
DNA?
2012). Recently,
it hason
beenBF?
shown
thatingenomic
DNA can be
present in the
blastocoele of the
human embryo, and that this DNA could be suitable for PCR, WGA and arrayCGH (Palini et al.,
C. PGD on human embryos: point mutation (MTHFR)
2013). However, the effective use of this DNA for PGD still needs to be demonstrated.
To further confirm that DNA found in blastocoele fluid samples was not derived from an external
contamination and to furnish a proof of concept regarding the possibility to detect a point mutation
from blastocoele fluid DNA, we attempted to genotype the methylenetetrahydrofolate reductase
(MTHFR) C677T polymorphism (Ueland et al., 2001) in blastocoele fluid sample from blastocysts
derived from an IVF patient known to carry the 677T mutation in homozygosis. To this end, WGA
has been attempted, followed by PCR and direct sequencing, or HRM analysis.
4. PGD on BF? DNA in BF represent embryo’s DNA?
C. PGD on human embryos: point mutation (MTHFR)
1. MTHFR amplification and sequencing successful in 6 out of 9 fluid samples (66.6%)
2. The WGA is not purify but directed tested
CONCLUSIONS
4. PGD on BF? DNA in BF represent embryo’s DNA?
ANSWER TO THE FOURTH QUESTION
1. GOOD IDEA!
• The sex of equine and human embryos can be
diagnosed using blastocele fluid as DNA source
for conventional PCR
• Is possible genotype point mutation in BF
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24.02.2015
Prof. L. Zolla
Department of Molecular
Biology and Proteomics,
University of la Tuscia, Viterbo
(VT), Italy
CONCLUSIONS
2.
THERE IS HUMAN GENOMIC DNA IN BF AND REPRESENT EMBRYO’S DNA
3.
BF’S DNA IS FRAGMENTED, APOPTOSIS FOR REGULATION OF CELLS NUMBER AND
“LINES” OR FOR REGULATORY MECHANISM UNKNOW RIGHT NOW?
2.
PROBABLY DNA FROM BF DERIVES BY BOTH ICM AND TE, GIVING A COMPLETE
INFORMATION ABOUT CHROMOSOME STATUS OF THE ENTIRE EMBRYO, PGS IS A
PHOTO OF THE MOMENT
2.
IN THE FUTURE PROBABLY WE WILL USE BF TO KNOW EMBRYO VIABILITY
3.
WE MUST THINK ABOUT THE BF AS A CULTURE MEDIUM FOR EMBRYO INTRA/EXTRA
COMMUNICATION
WIDER IMPLICATIONS OF THE FINDINGS:
• THE USE OF CELL-FREE gDNA IN MEDIUM OR BLASTOCOELE FLUID MAY
PROVIDE A NOVEL, NON-INVASIVE APPROACH FOR EMBRYO GENOTYPING
2012
2010
Submission to
ethical committee
2011
Ethical approval
2012
first sample
analysis
A mass spectrometry
based targeted
metabolomics strategy of
human BF: a promising
tool in infertility research
2013
Genomic DNA in Human
BF article
Prof. M. Magnani
Prof. L. Galluzzi
Department of Biomolecular
Sciences, University of Urbino ‘Carlo
Prof. Dagan Wells
Oxford Uk
Bo’, Urbino (PU), Italy
Dott. Simone Palini
Senior clinical embryologist
O.U. Physiopathology of Reproduction
Cervesi Hospital-Cattolica (RN) ITALY
[email protected]
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