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ORIGINAL ARTICLE: GENETICS 
Blastocentesis: a source of DNA for 
preimplantation genetic testing. 
Results from a pilot study 
Luca Gianaroli, M.D., M. Cristina Magli, M.Sc., Alessandra Pomante, Ph.D., Anna M. Crivello, B.Sc., 
Giulia Cafueri, B.Sc., Marzia Valerio, B.Sc., and Anna P. Ferraretti, M.D. 
Reproductive Medicine Unit, Societa Italiana Studi di Medicina della Riproduzione, Bologna, Italy 
Objective: To investigate the presence of DNA in blastocyst fluids (BFs) and to estimate whether the chromosomal status predicted by 
its analysis corresponds with the ploidy condition in trophectoderm (TE) cells, the whole embryo, and that predicted by polar bodies 
(PBs) or blastomeres. 
Design: Prospective study. 
Setting: In vitro fertilization unit. 
Patient(s): Seventeen couples undergoing preimplantation genetic screening with the use of array comparative genomic hybridization 
on PBs (n ¼ 12) or blastomeres (n ¼ 5). 
Intervention(s): BFs and TE cells were retrieved from 51 blastocysts for separate chromosomal analysis. 
Main Outcome Measure(s): Presence of DNA in BFs and assessment of the corresponding chromosome condition; correlation with the 
results in TE cells and those predicted by the analysis done at earlier stages. 
Result(s): DNA was detected in 39 BFs (76.5%). In 38 of 39 cases (97.4%) the ploidy condition of BFs was confirmed in TE cells, and the 
rate of concordance per single chromosome was 96.6% (904/936). In relation to the whole embryo, the ploidy condition corresponded in 
all cases with a per-chromosome concordance of 98.1%. The testing of PBs and blastomeres had 93.3% and 100% prediction of BF 
ploidy condition with a concordance per chromosome of 93.5% and 94%, respectively. 
Conclusion(s): Blastocentesis could represent an alternative source of material for chromosomal testing, because the BF is highly 
predictive of the embryo ploidy condition and chromosome content. Our data confirm the rele-vance 
of the oocyte and of the early-cleavage embryo in determining the ploidy condition of the 
resulting blastocyst. (Fertil Steril 2014;-:-–-. 2014 by American Society for 
Reproductive Medicine.) 
Key Words: Blastocyst, blastocele, blastomere, polar bodies, preimplantation genetic screening 
Discuss: You can discuss this article with its authors and with other ASRM members at http:// 
fertstertforum.com/gianarolil-blastocentesis-preimplantation-genetic-testing/ 
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The first sign of morphologic dif-ferentiation 
in the mammalian 
embryo happens at the morula 
stage, when some cells are diverted to 
become part of the inner cell mass 
(ICM) and others to generate the tro-phectoderm 
(TE) lineage, organized as 
an outer layer surrounding the ICM. 
After the formation of the two lin-eages, 
Naþ ions are accumulated on the 
basolateral side of the TE epithelium, 
creating an osmotic gradient that is 
partially originated by different iso-forms 
of Naþ/Kþ adenosine triphos-phatases 
(1). This gradient promotes 
the accumulation of water across the 
epithelium through the activity of 
transmembrane channels (2). The accu-mulated 
fluid merges and expands as a 
single entity, forming the cavity known 
as the blastocele. The subsequent devel-opment 
of tight junctions makes a seal, 
preventing leakage of the liquid from 
the cavity (3). These processes in the 
human embryo normally happen 4– 
5 days after fertilization, when the blas-tocyst 
stage begins. The progressive 
accumulation of the fluid inside the 
cavity and the constant duplication of 
cells lead to the enlargement of the 
blastocyst and the thinning of the 
zona pellucida (ZP). These steps culmi-nate 
in the hatching of the blastocyst 
from a natural breach in the ZP. At 
this stage, in vivo, the embryo is ready 
to implant in a receptive uterus. 
In assisted reproductive technol-ogy, 
culture to blastocyst has been pro-posed 
as a method for an efficient 
selection of the best embryo to transfer. 
Nevertheless, top-quality embryos, 
Received June 8, 2014; revised and accepted August 13, 2014. 
L.G. has nothing to disclose. M.C.M. has nothing to disclose. A.P. has nothing to disclose. A.M.C. has 
nothing to disclose. G.C. has nothing to disclose. M.V. has nothing to disclose. A.P.F. has nothing 
to disclose. 
Reprint requests: Luca Gianaroli, M.D., Reproductive Medicine Unit, Societa Italiana Studi di Medicina 
della Riproduzione, V. Mazzini 12, 40138 Bologna, Italy (E-mail: luca.gianaroli@sismer.it). 
Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 
Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. 
http://dx.doi.org/10.1016/j.fertnstert.2014.08.021 
VOL. - NO. - / - 2014 1
ORIGINAL ARTICLE: GENETICS 
including blastocysts, frequently fail to implant, mainly 
because of aneuploidy which is recognized as one of the 
main factors affecting embryo implantation (4–8). For this 
reason, preimplantation genetic screening (PGS) techniques, 
through the analysis of the embryo chromosome status, 
should help in improving the pregnancy rates by avoiding 
the transfer of aneuploid embryos. 
According to recently published data, blastocysts 
represent the stage providing the most reliable results for 
PGS (9–11). Biopsy at earlier stages, polar bodies (PBs) and 
blastomeres, seems to be inadequate owing to the additional 
abnormalities contributed by sperm and initial mitoses (for 
PB testing), and to mosaicism (especially for blastomere 
analysis). Biopsy of TE cells has several advantages: More 
than one cell is available, the embryonic mass is not 
touched, and the results obtained provide the most 
complete representation of the preimplantation embryo's 
chromosomal constitution. At this stage, correction 
mechanisms of meiotic reciprocal aneuploidies, if any, have 
already occurred, preventing the exclusion from transfer of 
blastocysts resulting from meiotic aneuploidy rescue (12). 
Though to a lower extent compared with the embryo at the 
cleavage stage, mosaicism can still be detected in 
blastocysts (4, 13). Therefore, even if it is not clear to what 
extent TE cells are representative of the ICM, clinical results 
of the application of PGS on blastocysts seem to be 
reassuring and very promising (9–11). 
Very recently, it was reported that the blastocyst fluid 
(BF) contains DNA, possibly representing another source 
of DNA for genetic analysis (14). Should these findings be 
confirmed, the aspiration of the BF, a procedure that we 
term blastocentesis, could easily become the preferred 
means of blastocyst biopsy. The aims of the present study 
were: 1) to verify the presence of DNA in BFs; 2) to estimate 
whether the chromosomal status predicted by its analysis 
corresponds with the ploidy condition of PBs, blastomeres, 
and TE cells; and 3) to estimate whether the chromosomal 
status predicted by PBs, blastomeres, BFs, and TE cell anal-ysis 
corresponds with the ploidy condition of the whole 
embryo. 
MATERIALS AND METHODS 
Plan of the Study 
This was a prospective study including 17 couples (maternal 
age 37.6  3.5 y) undergoing PGS by array comparative 
genomic hybridization (CGH) in PBs (12 patients) or blasto-meres 
(5 patients) because of advance maternal age (n ¼ 14) 
or repeated IVF failures (n ¼ 3). In all, these couples generated 
71 blastocysts, of which 51 were investigated for the presence 
of DNA in the BF. 
Each of the patients signed an informed consent to have 
further chromosomal analysis performed on supernumerary 
embryos. Identification of these embryos took into consider-ation 
the following aspects: 
 Development to blastocyst. 
 Aneuploid status predicted by chromosome assessment 
performed on PBs or blastomeres. 
 Euploid status predicted by chromosome assessment 
performed on PBs or blastomeres in blastocysts destined 
to cryopreservation. 
According to the study plan, the BF was aspirated and 
TE cells were biopsied for separate chromosomal analysis. 
When discarded from possible clinical use, and after the BF 
and TE biopsies were performed, whole blastocysts classified 
as nonviable (15) also underwent chromosomal analysis. 
The study was approved by our Institutional Review 
Board (IRB no. 20110503). 
Biopsy Procedures 
Biopsy of PB1 and PB2 was sequentially performed immedi-ately 
before intracytoplasmic sperminjection (ICSI; PB1 biopsy) 
and 6–9 hours after ICSI (PB2 biopsy). The ZP was opened me-chanically 
after meticulous removal of all adhering cumulus 
cells. PBs were transferred to polymerase chain reaction (PCR) 
tubes to be processed separately for chromosomal analysis (16). 
Blastomere biopsy was performed at 62 hours after ICSI 
in embryos at the 6–8-cell stage presenting with regular 
morphology (15). An infrared diode laser was used to open 
a small breech in the ZP from where all adhering cumulus 
cells had been removed. A nucleated blastomere was gently 
aspirated, transferred to a PCR tube, and processed for chro-mosome 
analysis. 
BFs from expanded blastocysts were aspirated by an ICSI 
pipette that was inserted in the point of contact between two 
TE cells to minimize the amount of crossed cytoplasm 
(Supplemental Fig. 1, available online at www.fertstert.org). 
Great attention was paid to avoid the aspiration of any 
cellular material. The aspirated fluid was directly transferred 
to a PCR tube kept on ice without the addition of any buffer. 
The tubes were immediately spun and stored at 80C until 
further processing. A single aspiration was performed, and 
the volume of the aspirated fluid was 1 mL. 
TE biopsy was performed by removing 3–5 cells that had 
herniated through the breach previously opened in the ZP at 
the time of PB or blastomere biopsy. When necessary, the bi-opsy 
procedure was completed by laser pulses. The retrieved 
cells were transferred to a PCR tube for further processing. 
In case of blastocysts not considered for clinical use (15), 
the whole embryo, after the above biopsies, was transferred to 
a PCR tube and processed. 
All biopsies were performed in Hepes-buffered medium 
supplemented with protein supplement (5 mg protein/mL) 
under oil (Lifeglobal Media). The PCR tubes containing the 
biopsies were stored at 80C until further processing. 
Whole-genome Amplification and Array 
Comparative Genomic Hybridization 
Amplification of all biopsies was performed in a class II 
laminar flow cabinet with the use of the Sureplex kit 
(Rubicon; Bluegnome). The quality of the DNA amplification 
was determined by loading 5 mL of the final reaction onto a 
1.5% agarose gel. An aliquot of the amplified DNA was 
labeled for array CGH (24sure; Bluegnome). After array 
CGH, each sample was analyzed for the presence of gains 
2 VOL. - NO. - / - 2014
and losses with the use of Blue-Fuse Multi software and the 
euploid/aneuploid status of the corresponding oocyte or em-bryo 
predicted (17). Visualization and reporting of aneuploidy 
was on a per-chromosome basis. 
For the evaluation of the results from the different stages, 
two indicators were used: the ploidy condition and the chro-mosome 
concordance. The first defines the correspondence 
among the different stages of the studied embryo in terms 
of euploidy or aneuploidy. This indicator has a clinical 
impact, because an embryo is assessed as transferrable or 
not transferrable based on its ploidy condition. The term chro-mosome 
concordance defines the percentage of correspon-dence 
of all studied chromosomes between the different 
stages of the analyzed embryos. Full concordance indicates 
cases where the ploidy condition was confirmed and all single 
chromosomes corresponded. Partial concordance refers to 
cases where the ploidy condition was confirmed, but not all 
single chromosomes corresponded. Null concordance in-cludes 
cases where the ploidy condition was discordant. 
RESULTS 
The patients included in the study generated 71 blastocysts, 
corresponding to a 69% blastocyst rate calculated per number 
of fertilized oocytes. This figure was similar after PB and blas-tomere 
biopsy. The chromosome status of the embryos that 
failed to develop to blastocysts was mostly aneuploid (74%). 
The BF was aspirated from 51 blastocysts, of which 37 
were from oocytes having had PB biopsy and 14 from 
embryos biopsied at the cleavage stage. 
Analysis of the Blastocelic Fluid for the Presence of 
DNA 
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, 
Fertility and Sterility® 
and TE cells (Supplemental Tables 1 and 2, available online at 
www.fertstert.org). The mean amount of DNA detected, 
amplified from the BF, was 900.38 ng/mL (range 876.3– 
939.5 ng/mL). 
In the 12 BF samples with no results (23.5%), failed ampli-fication 
occurred, as revealed by the absence of a specific 
band in the agarose gel run for 5 minutes. However, when 
the gel was electrophoresed for a longer time, a smear 
appeared, suggesting possible DNA degradation in the orig-inal 
sample (lane 6 in Supplemental Fig. 2, available online 
at www.fertstert.org). The subsequent labeling and hybridiza-tion 
led to inconclusive results. Ten of these blastocysts 
derived from aneuploid oocytes (n ¼ 6) or blastomeres 
(n ¼ 4), whereas the remaining two were predicted to be 
euploid by PB (n ¼ 1) or blastomere (n ¼ 1) testing. The whole 
embryo could be analyzed in seven cases, and the results 
confirmed those obtained in the previous biopsies. 
Chromosomal Complement of the Blastocelic Fluid 
in Relation to the Data Obtained by Polar Bodies or 
Blastomere and Trophectoderm Cells 
The data from the 39 BFs with informative results were eval-uated 
in relation to the data obtained with the use of the chro-mosomal 
testing in PBs (n ¼ 30) or blastomeres (n ¼ 9; 
Table 1). In all, BFs reflected the ploidy condition predicted 
by PBs in 93.3% of cases (28/30) and by blastomeres in 
100% of cases (9/9), accounting for a total ploidy concor-dance 
of 94.9% of cases (37/39), with two cases (5.1%) 
discordant. 
In more detail, for PBs there was full chromosome 
concordance in 21 samples (70% of 30), partial concordance 
in seven samples (23.3%), and null concordance in the re-maining 
two samples (6.7%). For blastomeres, full chromo-some 
concordance was found in eight samples (88.9% of 9) 
and partial concordance in one sample (11.1%). 
TABLE 1 
Overall chromosome concordance by stage at biopsy calculated per ploidy condition (euploid vs. aneuploid) and per single chromosome. 
Concordance, n (%) 
Full Partial Null Total 
BFs vs. PB 
Embryos 21 (70) 7 (23.3) 2 (6.7) 30 
Chromosomes 483/483 (100) 126/161 (78.3) 36/46 (78.3) 645/690 (93.5)a 
BFs vs. blastomeres 
Embryos 8 (88.9) 1 (11.1) 0 9 
Chromosomes 192/192 (100) 11/24 (46) 203/216 (94.0) 
BFs vs. TE cells 
Embryos 32 (82) 6 (15.4) 1 (2.6) 39 
Chromosomes 768/768 (100) 121/144 (84.0) 15/24 (62.5) 904/936 (96.6)a 
TE cells vs. PBs 
Embryos 21 (70) 8 (26.7) 1 (3.3) 30 
Chromosomes 483/483 (100) 152/184 (82.6) 22/23 (95.7) 657/690 (95.2) 
TE cells vs. blastomeres 
Embryos 7 (77.8) 2 (22.2) 0 9 
Chromosomes 168/168 (100) 38/48 (79.2) 206/216 (95.4) 
Note: BF ¼ blastocyst fluid; PBs ¼ polar bodies; TE ¼ trophectoderm. 
a P.01. 
Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 
VOL. - NO. - / - 2014 3
ORIGINAL ARTICLE: GENETICS 
Altogether, a full chromosome concordance was detected 
in 29 BF samples with the corresponding PBs (n ¼ 21) and 
blastomeres (n ¼ 8) (Supplemental Table 1). A euploid condi-tion 
had been predicted in six cases (three after PB testing and 
three after blastomere testing) and confirmed in the BF of the 
resulting blastocysts, including one case where all the 
aneuploidies detected in PB1 were compensated by reciprocal 
aneuploidies in PB2 (Fig. 1). The remaining 23 samples were 
confirmed to be aneuploid (see an example in Fig. 2). 
Partial concordance was detected in 8 samples, which 
were all predicted to be aneuploid by PBs (n ¼ 7) or blasto-mere 
(n ¼ 1). Here, the ploidy condition was concordant, 
but BFs showed aneuploidies that were only partially pre-dicted 
by the analysis at the previous stages (Supplemental 
Table 2). An example is shown in Supplemental Figure 3 
(available online at www.fertstert.org). 
Null concordance was found in two samples, both defined 
as aneuploid by PB testing. Here, the ploidy condition found 
in the BFs was discordant with the prediction made by PBs 
(Supplemental Table 2). In one of these cases, chromosomal 
abnormalities were found only in the BF, whereas PBs and 
TE cells were euploid. In the other case, PBs predicted an em-bryo 
trisomic for chromosome 6, but the resulting blastocyst 
was euploid according to the analysis of BF, TE cells, and the 
whole embryo. 
When comparing the results from BFs and the corre-sponding 
TE cells, in 97.4% of sets (38/39) the ploidy condi-tion 
of BFs was confirmed in TE cells (Table 1). In more 
detail, there was full chromosome concordance in 32 cases 
(82% of 39), partial concordance in six cases (15.4%), and 
null concordance in one case (2.6%). The discordant 
case was classified as aneuploid by BF analysis and 
euploid by TE analysis with nine discordant chromosomes 
(Supplemental Table 2). 
Looking at single chromosomes in the whole dataset, the 
rate of concordance between BF and the other biopsies was 
93.5% with PBs, 94% with blastomeres, and 96.6% with 
TE cells (96.6% vs. 93.5%; P.01; Table 1). 
No statistically significant difference was detected when 
the results from TE cell analysis were compared with the 
data obtained by PBs and blastomeres (Table 1). 
Chromosomal Complement of the Blastocyst 
Assessed by the Analysis of the Whole Embryo in 
Relation to the Data Obtained by Polar Bodies, 
Blastomeres, Blastocyst Fluid, and Trophectoderm 
Cells 
To add further information regarding the comparisons of the 
chromosome results obtained at each stage, the whole embryo 
was analyzed in 26 sets, 20 having had PB biopsy and six blas-tomere 
biopsy (Table 2; Supplemental Table 3, available online 
at www.fertstert.org). All of them had had BF and TE biopsies. 
The ploidy condition of the whole embryo was predicted 
in 95% of cases by PBs (19/20), in 100% of cases by blasto-meres 
(6/6), and in 100% of cases by both BF and TE cells 
(26/26; Table 2). The only discordant case was predicted as 
aneuploid for chromosome 6 by PBs and found to be euploid 
in the whole embryo as well as in the corresponding BF and 
TE cells (Supplemental Table 3). 
Full chromosome concordance was similar throughout all 
stages, namely, 80% with PBs (16/20), 100% with blastomeres 
(6/6), 80.8% with TE cells (21/26), and 84.6% with BF (22/26). 
Partial concordance was 15% with PBs (3/20), 19.2% with 
FIGURE 1 
The aneuploidies in polar body 1 (PB1) were compensated by reciprocal aneuploidies in polar body 2 (PB2). As a result, the corresponding blastocyst 
was euploid according to analysis of the blastocelic fluid (BF) and trophectodem cells (TE). 
Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 
4 VOL. - NO. - / - 2014
TE cells (5/26), and 15.4% with BFs (4/26). Finally, null 
concordance was found with one PB case (5% of 20). 
Calculated per single chromosome, the grade of concor-dance 
was 98.9% with PBs, 100% with blastomeres, 97.9% 
with BFs, and 98.1% with TE cells. 
DISCUSSION 
The most relevant findings in our study were the detection of 
DNA in the majority of BFs and the strong prediction value of 
PB and blastomere testing on the blastocyst chromosomal 
status. 
In 23.5% of the investigated BFs, no informative DNA 
could be detected. No morphologic factors in the studied blas-tocysts 
could be recognized as able to predict the finding of 
DNA in BFs, although a common feature in the blastocysts 
with failed amplification was the presence of a poor-quality 
TE. On the other hand, this characteristic did not prevent 
the finding of DNA in the BFs of other cases. After attempting 
WGA, we observed that the samples that failed to amplify 
probably had highly fragmented DNA, as suggested by the 
smear observed in the agarose gel when it was electrophor-esed 
for 15 minutes (Supplemental Fig. 2). More cases are 
needed to see if there is a connection between these observa-tions 
and the absence of informative DNA in the BF. Technical 
problems could also have occurred, especially during the 
tubing of the BF, and this is a step that we are carefully revis-iting. 
We are also trying to evaluate if the time of biopsy could 
have an effect on the subsequent outcome. As a preliminary 
finding, we retrieved the BF twice from two blastocysts at 
24-hour intervals. In one case, WGA failed in the first biopsy 
and was successful in the second sample; in the other case, 
Fertility and Sterility® 
both samples were successful and provided the same 
diagnosis. 
The presence of DNA in BFs, detected and successfully 
tested in 76.5% of the studied samples, is actually not surpris-ing. 
All cells surrounding the blastocele are metabolically 
active and secrete different substances, including several 
types of proteins (18, 19). It is well known that cell death 
normally occurs even in good-quality blastocysts, in both 
ICM and TE compartments. A study evaluating blastocysts 
originating from embryos with different levels of fragmenta-tion 
demonstrated higher levels of apoptosis in embryos of 
excellent morphology, suggesting a possible role of apoptosis 
in the regulation of cell number (20). Because the aspiration of 
the fluid was done by carefully avoiding the contamination 
by any cellular material, we are confident that the extracted 
DNA is a true reflection of the BF content. In addition, the 
detection of some differences in the chromosome status of 
BFs compared with that of TE cells supports a different origin 
of the two samples. 
Based on the above considerations, the presence of DNA 
in BFs could be consequent to its release from dead cells into 
the blastocelic cavity, with its actual quantity being possibly 
related to the rate of cell death. Therefore, there could be 
three different scenarios in the analysis of the BF: 1) The 
DNA in the BF is too fragmented or too scarce to result in 
successful genome amplification; 2) the DNA in the BF am-plifies 
and appears to be a reflection of the ploidy condition 
of the blastocyst, taking TE cells as the reference (TE biopsy 
being the conventional procedure for PGS of blastocysts); 
and 3) the DNA in the BF is discordant with the chromo-somal 
complement of the blastocyst, taking TE cells as the 
reference. 
FIGURE 2 
A gain for chromosome 8 in PB1 and for chromosomes 15 and 22 in PB2 predicted a blastocyst monosomic for chromosomes 8, 15, and 22. This 
condition was confirmed in both the BF and the TE cells. Abbreviations as in Figure 1. 
Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 
VOL. - NO. - / - 2014 5
ORIGINAL ARTICLE: GENETICS 
Regarding the first scenario, this happened in our dataset 
at a frequency of 23.5%, with no informative DNA found in 
12 of the 51 studied samples. As already mentioned, no fac-tors 
were identified that could be associated with the 
negative or positive finding of DNA. 
For the second scenario, the analysis of the BF was in 
agreement with that of the corresponding TE cells in 38 
samples (74.5% of 51 samples) with full chromosome 
concordance per single chromosome in 32 of them. 
Finally, the remaining set represented the third scenario 
(2% of 51 samples), with BF showing multiple chromosome 
anomalies in a blastocyst that was predicted to be euploid 
by PB and TE analysis (Table 1). 
In summary, the ploidy condition between BFs and 
TE cells was found to be concordant in all cases except one 
(38/39, 97.4%), suggesting that BF could represent a valuable 
material for clinical use, provided that the proportion of sam-ples 
with informative DNA is raised to levels similar to those 
obtained by conventional biopsy procedures. 
Looking at single chromosomes, the global evaluation of 
the data in the 39 samples with successful amplification and 
diagnosis of DNA in the BF demonstrated full concordance 
between BFs and TE cells in 82% of cases (32/39), with the 
DNA retrieved by blastocentesis reflecting the exact chromo-somal 
content of TE cells. In the remaining sets, some chro-mosome 
variations were detected between BFs and TE cells, 
which is not surprising if we consider the possible origin of 
DNA in BF, which could also contain DNA released from 
the ICM (20). As presented in Table 1, the BF was extremely 
sensitive, similarly to TE cells, in revealing all of the chromo-some 
variations that occurred during embryo development. 
As expected, the concordance per single chromosome was 
significantly lower with PBs compared with TE cells, confirm-ing 
that the BF collects the DNA produced by TE cells as well 
as the ICM. 
In view of our results, because the artificial collapse of 
the blastocele is routinely applied in the vitrification of 
expanded and hatching blastocysts (21), it would be advis-able 
to store the BF and to have it at least as a back-up 
sample until further refinement of its use as alternative 
DNA source is reached. 
The second relevant finding coming from our study is the 
high level of prediction of the blastocyst chromosomal status 
made by the analysis done at the previous stages. Taking TE 
cells as the reference (Table 1), the prediction of ploidy 
made by PB or blastomere testing was confirmed in 97.4% 
of cases (96.7% and 100%, respectively). When considering 
the number of abnormalities per single chromosome, PBs 
and blastomeres had similar prediction power of the chromo-some 
complement of TE cells (95.2% and 95.4% respectively). 
A similar trend was observed when taking the whole embryo 
as the reference (Table 2), with the ploidy condition confirmed 
in 95% of PBs and 100% of blastomeres but the concordance 
per single chromosome rising to 98.9% and 100%, respec-tively. 
In other words, TE cells are only partially representa-tive 
of the whole blastocysts, because the ICM could have 
some differences regarding chromosome condition. It can 
be speculated that the BF collecting DNA from both TE and 
ICM could be more sensitive in defining the whole blastocyst 
chromosome status. The high prediction level of PBs of the 
whole embryo chromosome condition is in contrast with 
other authors claiming that testing at the PB stage is the 
less accurate method compared with blastomeres, mainly 
owing to the high incidence of post-zygotic events (22). In 
this respect, it must be mentioned that in that work the 
authors assumed that TE cells are representative of the blasto-cyst 
chromosome complement, an assumption that they 
corroborated in another work with the use of fluorescence 
in situ hybridization (23). This is probably true in most cases, 
especially when considering our data as well as the positive 
clinical outcome associated with PGS on blastocysts (10, 11, 
24). However, when we analyzed the whole embryo and 
compared the results with those derived from TE cells, the 
concordance per single chromosomes was 97.9% (Table 2; 
Supplemental Table 3). Based on these findings, taking 
TE cells as the point of reference to evaluate the power of 
prediction of PGS made at previous stages could be not 
totally correct. On the other hand, the capacity of BF in 
TABLE 2 
Overall chromosome concordance by stage at biopsy over the whole embryo calculated per ploidy condition (euploid vs. aneuploid) and per single 
chromosome. 
Concordance, n (%) 
Full Partial Null Total 
Whole embryo vs. PBs 
Embryos 16 (80) 3 (15) 1 (5) 20 
Chromosomes 368/368 (100) 65/69 (94.2) 22/23 (95.7) 455/460 (98.9) 
Whole embryo vs. blastomere 
Embryos 6 (100) 0 0 6 
Chromosomes 144/144 (100) 144/144 (100) 
Whole embryo vs. TE cells 
Embryos 21 (80.8) 5 (19.2) 0 26 
Chromosomes 504/504 (100) 107/120 (89.2) 611/624 (97.9) 
Whole embryo vs. BF 
Embryos 22 (84.6) 4 (15.4) 0 26 
Chromosomes 528/528 (100) 84/96 (87.5) 612/624 (98.1) 
Note: Abbreviations as in Table 1. 
Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 
6 VOL. - NO. - / - 2014
defining the whole embryo ploidy condition was 100% and 
98.1% per single chromosome, suggesting that an 
informative BF represents a true reflection of the blastocyst 
chromosome condition. Blastocentesis being a less invasive 
method of biopsy, this strategy could become the preferred 
choice for PGS. 
We observed that the lowest rates of concordance in BFs 
per single chromosome were frequently related to the pres-ence 
of multiple abnormalities in PBs or blastomeres. It 
could be speculated that highly aneuploid zygotes or early 
embryos are predisposed to develop mosaic blastocysts, 
possibly owing to defective sister chromatid cohesion that 
can result in chromosome missegregation and aneuploidy 
(25). Misaligned chromosomes in a metaphase aneuploid 
cell or lagging chromosomes between anaphase cells also 
promote the formation of mosaicism. It has been reported 
that aneuploidy induces an increase in DNA recombination 
and a decrease in DNA damage repair efficiency, which are 
both forms of genomic instability (26). In other words, aneu-ploidy 
per se seems to be able to induce chromosomal insta-bility, 
and this could explain the type of results obtained in 
some cases of partial concordance (Supplemental Table 2). 
More on the subject is added by an experimental mouse 
model showing that mosaic aneuploid embryos can develop 
and implant in female uterine tissue and initiate the gastru-lation 
process, but quickly degenerate (27). Searching for the 
mechanism responsible for the demise of these embryos, it 
was found that it is caused by the activation of a spatially 
and temporally controlled p53-independent apoptotic mech-anism 
and does not result from a failure to progress through 
mitosis (27). Therefore, the initial state of primary aneu-ploidy 
resulted in a rapid evolution of mosaicism and early 
embryonic death. This gestational loss due to aneuploid 
mosaicism could account for the large proportion of human 
pregnancy loss before clinical recognition. The per-chromosome 
discordances detected in our study between 
BFs and TE cells, and between them and the whole embryo, 
could be a reflection of this mechanism which could be 
especially relevant in the case of oocytes carrying multiple 
aneuploidies (Supplemental Table 2). 
In conclusion, the present data confirmed the relevance of 
the oocyte and of the early-cleavage embryo in determining 
the ploidy of the resulting blastocyst. Therefore, although 
the incidence of post-zygotic events can not be disregarded, 
the oocyte and the initial stages of embryogenesis do provide 
reliable results for PGS (and preimplantation genetic diag-nosis). 
The finding of DNA in BFs, besides representing a 
possible alternative source of material for chromosomal anal-ysis, 
could contribute additional information on the study of 
chromosome segregation in the early embryo. 
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et al. Embryos whose polar bodies contain isolated reciprocal chromo-some 
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ease/sister-chromatid-cohesion-and-aneuploidy. 
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Aneuploidy drives genomic instability in yeast. Science 2011;333:1026–30. 
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mosaic aneuploid embryos during mouse development. Dev Biol 2006; 
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8 VOL. - NO. - / - 2014
SUPPLEMENTAL FIGURE 1 
Aspiration of the blastocyst fluid from an expanded blastocyst. An 
intracytoplasmic sperm injection pipette was inserted in the point of 
contact between two trophectoderm cells to minimize the amount 
of crossed cytoplasm. Attention was paid to avoid the aspiration of 
cellular material. 
Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 
Fertility and Sterility® 
VOL. - NO. - / - 2014 8.e1
ORIGINAL ARTICLE: GENETICS 
SUPPLEMENTAL FIGURE 2 
Amplification product of seven blastocelic fluid (BF) samples on 1.5% 
agarose gel. (A) Gel electrophoresed for 5 minutes at 100 V, and (B) 
the same gel run for 15 minutes. The lane contents are labeled as 
follows: one kb ladder, followed by seven BF samples. The last two 
lanes are positive and negative control samples, respectively. The 
amplification band is strong in all samples and absent in sample 6 
(A). When the gel was run for 15 minutes, a smear appeared in 
sample 6 (B). 
Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 
8.e2 VOL. - NO. - / - 2014
SUPPLEMENTAL FIGURE 3 
Fertility and Sterility® 
A gain for chromosome 16 in the polar body 2 (PB2) predicted a blastocyst monosomic for chromosome 16. This condition was found in both the 
blastocelic fluid (BF) and trophectoderm cells (TE), but with the addition of monosomy 9. 
Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 
VOL. - NO. - / - 2014 8.e3
ORIGINAL ARTICLE: GENETICS 
SUPPLEMENTAL TABLE 1 
Chromosomal status in blastocelic fluid (BF) samples showing full correspondence with the results predicted by polar bodies (PBs) or 
blastomeres. 
Sample 
ID Age (y) PB1 PB2 Blastomere BF TE 
3 38 euploid loss 2 – gain 2 gain 2 
5 38 gain 8 gain 15, 22 – loss 8,15,22 loss 8,15,22 
7 36 – loss 14 loss 14 loss 14 
9 36 – euploid euploid euploid 
13 36 gain 4, 5, 6, 7, 9, 11, 12, 
15, 19, 20, X 
loss 1, 2, 3, 8, 10, 13, 14, 
16, 18 
gain 1, 2, 3, 8, 10, 13, 14, 
16, 18 
loss 4, 5, 6, 7, 9, 11, 12, 15, 
19, 20, X 
– euploid euploid 
19 42 euploid euploid – euploid euploid 
21 38 – – euploid euploid euploid 
22 38 – – euploid euploid euploid 
24 41 – – loss 16 loss 16 loss 16 
28 42 euploid loss 21 – gain 21 gain 21, 
loss 1 
29 41 euploid euploid – euploid euploid 
33 35 euploid gain 15 – loss 15 loss 15 
57 41 euploid gain 19, 21 
loss 18, 22 
– gain 18, 22 
loss 19, 21 
gain 18, 22 
loss 19, 21 
58 41 euploid loss 15 – gain 15 gain 15 
85 36 loss 22 loss 14 – gain 14, 22 gain 14, 22 
86 36 gain 9q 
loss 9p, 15 
loss 9 – gain 9q, 15 gain 9q, 15 
87 36 gain 16 
loss 13 
loss 16 – gain 13 gain 13 
88 36 loss 15 gain 15 (chromosome) – loss 15 loss 15 
89 36 gain 4q (chromosome) gain 16 
loss 4q 
– loss 16, 4q loss 16, 4q 
90 36 euploid gain 13 – loss 13 loss 13 
111 37 – – loss 8, 16 loss 8, 16 loss 8, 16 
112 37 – – gain 2, 7, 21 
loss 10 
gain 2, 7, 21 
loss 10 
gain 2, 7, 21 
loss 10, 11, 16 
114 37 – – gain 8, 16 gain 8, 16 gain 8, 16 
116 40 gain 21 
loss 13 
gain 22 – gain 13 
loss 21, 22 
gain 13 
loss 21, 22 
118 40 gain 15, 21 gain 22 loss 15, 21, 22 gain 10 
loss 15, 21, 22 
119 40 gain 7q 
loss 4, 7p 
loss 7 – gain 4, 7p 
(chromosome) 
gain 4, 7p 
(chromosome) 
121 40 loss 9 euploid – gain 9 gain 9 
124 40 gain 18 
loss 16 
gain 16 
loss 18, 22 
– gain 22 gain 22 
126 40 gain 21 
loss 18 
gain 18 
loss 8 
– gain 8 
loss 21 
gain 8 
loss 21 
Note: In all samples except three (samples 28, 112, and 118) the full concordance also included cells biopsied from the trophectoderm (TE). All gains and losses observed in PBs were due to 
chromatid predivision except where indicated. 
Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 
8.e4 VOL. - NO. - / - 2014
SUPPLEMENTAL TABLE 2 
Fertility and Sterility® 
Chromosomal status in BF samples and corresponding TE showing partial or null correspondence with the results predicted by PBs or 
blastomeres. 
Sample 
ID Age (y) PB1 PB2 Blastomere BF TE 
1 41 loss 16 loss 12 – gain 12,16 
loss 15 
gain 12,16 
loss 15 
4 38 euploid gain 22 – loss 17,22 loss 17,22 
11 36 euploid gain 16 – loss 9, 16 loss 9, 16 
6 37 gain 9 
loss 1 
gain 1, 16, 22 
loss 9 
– gain 12 
loss 15, 16 
loss 16,22 
8 36 – – gain 5, 10, 17, 21 
loss 1, 6, 14, 15, 22 
gain 8, 13, 21 
loss 1, 5, 17, 18, X 
gain 21 
10 36 gain 1, 2, 4, 8, 11, 12, 13, 
15, 20, 22 
loss 3, 5, 9, 10, 16, 18, 19, 
21, X 
euploid – loss 12 loss 12 
18 42 gain 4, 19 
– 
gain 5 
loss 4 
– gain 2, 4, 8, 12, 20 
loss 5, 13, 14 
gain 2,8,17,18 
loss 10,13,19 
70 38 gain 20 
loss 21 
euploid – gain 16 
loss 20 
gain 16 
loss 20 
2 38 euploid euploid – gain 5, 8, 11, 12, 15, 19 
loss 3, 9, 16 
euploid 
117 40 loss 6, 22 gain 22 – euploid euploid 
Note: Partial concordance was found in eight sets. In the first three samples (samples 1, 4, and 11), BFs corresponded with the predictions made by PBs and blastomeres with the addition of new 
aneuploidies. In the subsequent five samples (samples 6, 8, 10, 18, and 70), only some of the anomalies predicted by PBs and blastomeres appeared in BFs, and new ones were detected. In two cases 
(samples 10 and 70), BFs and TE cells fully matched. Null concordance was detected in the last two samples (2 and 117), where the chromosomal status of the DNA retrieved by the BF did not match 
with that predicted by PBs. All gains and losses observed in PBs were due to chromatid predivision. Abbreviations as in Supplemental Table 1. 
Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 
VOL. - NO. - / - 2014 8.e5
SUPPLEMENTAL TABLE 3 
Chromosome status in PBs or blastomeres compared with the results obtained in TE cells and the whole embryo. 
Sample ID Age (y) PB1 PB2 Blastomere BF TE Whole embryo 
19 42 euploid euploid - euploid euploid euploid 
21 38 – – euploid euploid euploid euploid 
22 38 – – euploid euploid euploid euploid 
24 41 – – loss 16 loss 16 loss 16 loss 16 
33 35 euploid gain 15 – loss 15 loss 15 loss 15 
57 41 euploid gain 19,21 
loss 18,22 
– gain 18,22 
loss 19,21 
gain 18, 22 
loss 19, 21 
gain 18, 22 
loss 19,21 
58 41 euploid loss 15 – gain 15 gain 15 gain 15 
86 36 gain 9q 
loss 9p,15 
loss 9 – gain 9q,15 gain 9q, 15 gain 9q, 15 
87 36 gain 16 
loss 13 
loss 16 – gain 13 gain 13 gain 13 
88 36 loss 15 gain 15 (chromosome) – loss 15 loss 15 loss 15 
89 36 gain 4q (chromosome) gain 16 
loss 4q 
– loss 16,4q loss 16, 4q loss 16, 4q 
90 36 euploid gain 13 – loss 13 loss 13 loss 13 
111 37 – – loss 8, 16 loss 8, 16 loss 8, 16 loss 8, 16 
114 37 – – gain 8, 16 gain 8, 16 gain 8, 16 gain 8, 16 
116 40 gain 21 
loss 13 
gain 22 – gain 13 
loss 21, 22 
gain 13 
loss 21, 22 
gain 13 
loss 21, 22 
119 40 gain 7q 
loss 4, 7p 
loss 7 – gain 4, 7p (chromosome) gain 4, 7p (chromosome) gain 4, 7p (chromosome) 
121 40 loss 9 euploid – gain gain 9 gain 9 
124 40 gain 18 
loss 16 
gain 16 
loss 18, 22 
– gain 22 gain 22 gain 22 
126 40 gain 21 
loss 18 
gain 18 
loss 8 
– gain 8 
loss 21 
gain 8 
loss 21 
gain 8 
loss 21 
18 42 gain 4, 19 gain 5 
loss 4 
– gain 2, 4, 8, 12, 20 
loss 5, 13, 14 
gain 2, 8, 17, 18 
loss 10, 13, 19 
loss 5, 19 
28 42 euploid loss 21 – gain 21 gain 21 
loss 1 
gain 21 
70 38 gain 20 
loss 21 
euploid – gain 16 
loss 20 
gain 16 
loss 20 
gain 7 
loss 20 
85 36 loss 22 loss 14 – gain 14, 22 gain 14, 22 gain 4, 14, 22 
112 37 – – gain 2, 7, 21 
loss 10 
gain 2, 7, 21 
loss 10 
gain 2, 7, 21 
loss 10, 11, 16 
gain 2, 7, 21 
loss 10 
117 40 loss 6 (chromosome), 22 gain 22 – euploid euploid euploid 
118 40 gain 15,21 gain 22 – loss 15, 21, 22 gain 10 
loss 15,21,22 
gain 10 
loss 15,21,22 
Note: The samples 19 to 126 are those with full concordance throughout all the studied stages. All gains and losses observed in PBs were due to chromatid predivision except where indicated. Abbreviations as in Supplemental Table 1. 
Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 
8.e6 VOL. - NO. - / - 2014 
ORIGINAL ARTICLE: GENETICS

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  • 1. ORIGINAL ARTICLE: GENETICS Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study Luca Gianaroli, M.D., M. Cristina Magli, M.Sc., Alessandra Pomante, Ph.D., Anna M. Crivello, B.Sc., Giulia Cafueri, B.Sc., Marzia Valerio, B.Sc., and Anna P. Ferraretti, M.D. Reproductive Medicine Unit, Societa Italiana Studi di Medicina della Riproduzione, Bologna, Italy Objective: To investigate the presence of DNA in blastocyst fluids (BFs) and to estimate whether the chromosomal status predicted by its analysis corresponds with the ploidy condition in trophectoderm (TE) cells, the whole embryo, and that predicted by polar bodies (PBs) or blastomeres. Design: Prospective study. Setting: In vitro fertilization unit. Patient(s): Seventeen couples undergoing preimplantation genetic screening with the use of array comparative genomic hybridization on PBs (n ¼ 12) or blastomeres (n ¼ 5). Intervention(s): BFs and TE cells were retrieved from 51 blastocysts for separate chromosomal analysis. Main Outcome Measure(s): Presence of DNA in BFs and assessment of the corresponding chromosome condition; correlation with the results in TE cells and those predicted by the analysis done at earlier stages. Result(s): DNA was detected in 39 BFs (76.5%). In 38 of 39 cases (97.4%) the ploidy condition of BFs was confirmed in TE cells, and the rate of concordance per single chromosome was 96.6% (904/936). In relation to the whole embryo, the ploidy condition corresponded in all cases with a per-chromosome concordance of 98.1%. The testing of PBs and blastomeres had 93.3% and 100% prediction of BF ploidy condition with a concordance per chromosome of 93.5% and 94%, respectively. Conclusion(s): Blastocentesis could represent an alternative source of material for chromosomal testing, because the BF is highly predictive of the embryo ploidy condition and chromosome content. Our data confirm the rele-vance of the oocyte and of the early-cleavage embryo in determining the ploidy condition of the resulting blastocyst. (Fertil Steril 2014;-:-–-. 2014 by American Society for Reproductive Medicine.) Key Words: Blastocyst, blastocele, blastomere, polar bodies, preimplantation genetic screening Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/gianarolil-blastocentesis-preimplantation-genetic-testing/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace. The first sign of morphologic dif-ferentiation in the mammalian embryo happens at the morula stage, when some cells are diverted to become part of the inner cell mass (ICM) and others to generate the tro-phectoderm (TE) lineage, organized as an outer layer surrounding the ICM. After the formation of the two lin-eages, Naþ ions are accumulated on the basolateral side of the TE epithelium, creating an osmotic gradient that is partially originated by different iso-forms of Naþ/Kþ adenosine triphos-phatases (1). This gradient promotes the accumulation of water across the epithelium through the activity of transmembrane channels (2). The accu-mulated fluid merges and expands as a single entity, forming the cavity known as the blastocele. The subsequent devel-opment of tight junctions makes a seal, preventing leakage of the liquid from the cavity (3). These processes in the human embryo normally happen 4– 5 days after fertilization, when the blas-tocyst stage begins. The progressive accumulation of the fluid inside the cavity and the constant duplication of cells lead to the enlargement of the blastocyst and the thinning of the zona pellucida (ZP). These steps culmi-nate in the hatching of the blastocyst from a natural breach in the ZP. At this stage, in vivo, the embryo is ready to implant in a receptive uterus. In assisted reproductive technol-ogy, culture to blastocyst has been pro-posed as a method for an efficient selection of the best embryo to transfer. Nevertheless, top-quality embryos, Received June 8, 2014; revised and accepted August 13, 2014. L.G. has nothing to disclose. M.C.M. has nothing to disclose. A.P. has nothing to disclose. A.M.C. has nothing to disclose. G.C. has nothing to disclose. M.V. has nothing to disclose. A.P.F. has nothing to disclose. Reprint requests: Luca Gianaroli, M.D., Reproductive Medicine Unit, Societa Italiana Studi di Medicina della Riproduzione, V. Mazzini 12, 40138 Bologna, Italy (E-mail: luca.gianaroli@sismer.it). Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.08.021 VOL. - NO. - / - 2014 1
  • 2. ORIGINAL ARTICLE: GENETICS including blastocysts, frequently fail to implant, mainly because of aneuploidy which is recognized as one of the main factors affecting embryo implantation (4–8). For this reason, preimplantation genetic screening (PGS) techniques, through the analysis of the embryo chromosome status, should help in improving the pregnancy rates by avoiding the transfer of aneuploid embryos. According to recently published data, blastocysts represent the stage providing the most reliable results for PGS (9–11). Biopsy at earlier stages, polar bodies (PBs) and blastomeres, seems to be inadequate owing to the additional abnormalities contributed by sperm and initial mitoses (for PB testing), and to mosaicism (especially for blastomere analysis). Biopsy of TE cells has several advantages: More than one cell is available, the embryonic mass is not touched, and the results obtained provide the most complete representation of the preimplantation embryo's chromosomal constitution. At this stage, correction mechanisms of meiotic reciprocal aneuploidies, if any, have already occurred, preventing the exclusion from transfer of blastocysts resulting from meiotic aneuploidy rescue (12). Though to a lower extent compared with the embryo at the cleavage stage, mosaicism can still be detected in blastocysts (4, 13). Therefore, even if it is not clear to what extent TE cells are representative of the ICM, clinical results of the application of PGS on blastocysts seem to be reassuring and very promising (9–11). Very recently, it was reported that the blastocyst fluid (BF) contains DNA, possibly representing another source of DNA for genetic analysis (14). Should these findings be confirmed, the aspiration of the BF, a procedure that we term blastocentesis, could easily become the preferred means of blastocyst biopsy. The aims of the present study were: 1) to verify the presence of DNA in BFs; 2) to estimate whether the chromosomal status predicted by its analysis corresponds with the ploidy condition of PBs, blastomeres, and TE cells; and 3) to estimate whether the chromosomal status predicted by PBs, blastomeres, BFs, and TE cell anal-ysis corresponds with the ploidy condition of the whole embryo. MATERIALS AND METHODS Plan of the Study This was a prospective study including 17 couples (maternal age 37.6 3.5 y) undergoing PGS by array comparative genomic hybridization (CGH) in PBs (12 patients) or blasto-meres (5 patients) because of advance maternal age (n ¼ 14) or repeated IVF failures (n ¼ 3). In all, these couples generated 71 blastocysts, of which 51 were investigated for the presence of DNA in the BF. Each of the patients signed an informed consent to have further chromosomal analysis performed on supernumerary embryos. Identification of these embryos took into consider-ation the following aspects: Development to blastocyst. Aneuploid status predicted by chromosome assessment performed on PBs or blastomeres. Euploid status predicted by chromosome assessment performed on PBs or blastomeres in blastocysts destined to cryopreservation. According to the study plan, the BF was aspirated and TE cells were biopsied for separate chromosomal analysis. When discarded from possible clinical use, and after the BF and TE biopsies were performed, whole blastocysts classified as nonviable (15) also underwent chromosomal analysis. The study was approved by our Institutional Review Board (IRB no. 20110503). Biopsy Procedures Biopsy of PB1 and PB2 was sequentially performed immedi-ately before intracytoplasmic sperminjection (ICSI; PB1 biopsy) and 6–9 hours after ICSI (PB2 biopsy). The ZP was opened me-chanically after meticulous removal of all adhering cumulus cells. PBs were transferred to polymerase chain reaction (PCR) tubes to be processed separately for chromosomal analysis (16). Blastomere biopsy was performed at 62 hours after ICSI in embryos at the 6–8-cell stage presenting with regular morphology (15). An infrared diode laser was used to open a small breech in the ZP from where all adhering cumulus cells had been removed. A nucleated blastomere was gently aspirated, transferred to a PCR tube, and processed for chro-mosome analysis. BFs from expanded blastocysts were aspirated by an ICSI pipette that was inserted in the point of contact between two TE cells to minimize the amount of crossed cytoplasm (Supplemental Fig. 1, available online at www.fertstert.org). Great attention was paid to avoid the aspiration of any cellular material. The aspirated fluid was directly transferred to a PCR tube kept on ice without the addition of any buffer. The tubes were immediately spun and stored at 80C until further processing. A single aspiration was performed, and the volume of the aspirated fluid was 1 mL. TE biopsy was performed by removing 3–5 cells that had herniated through the breach previously opened in the ZP at the time of PB or blastomere biopsy. When necessary, the bi-opsy procedure was completed by laser pulses. The retrieved cells were transferred to a PCR tube for further processing. In case of blastocysts not considered for clinical use (15), the whole embryo, after the above biopsies, was transferred to a PCR tube and processed. All biopsies were performed in Hepes-buffered medium supplemented with protein supplement (5 mg protein/mL) under oil (Lifeglobal Media). The PCR tubes containing the biopsies were stored at 80C until further processing. Whole-genome Amplification and Array Comparative Genomic Hybridization Amplification of all biopsies was performed in a class II laminar flow cabinet with the use of the Sureplex kit (Rubicon; Bluegnome). The quality of the DNA amplification was determined by loading 5 mL of the final reaction onto a 1.5% agarose gel. An aliquot of the amplified DNA was labeled for array CGH (24sure; Bluegnome). After array CGH, each sample was analyzed for the presence of gains 2 VOL. - NO. - / - 2014
  • 3. and losses with the use of Blue-Fuse Multi software and the euploid/aneuploid status of the corresponding oocyte or em-bryo predicted (17). Visualization and reporting of aneuploidy was on a per-chromosome basis. For the evaluation of the results from the different stages, two indicators were used: the ploidy condition and the chro-mosome concordance. The first defines the correspondence among the different stages of the studied embryo in terms of euploidy or aneuploidy. This indicator has a clinical impact, because an embryo is assessed as transferrable or not transferrable based on its ploidy condition. The term chro-mosome concordance defines the percentage of correspon-dence of all studied chromosomes between the different stages of the analyzed embryos. Full concordance indicates cases where the ploidy condition was confirmed and all single chromosomes corresponded. Partial concordance refers to cases where the ploidy condition was confirmed, but not all single chromosomes corresponded. Null concordance in-cludes cases where the ploidy condition was discordant. RESULTS The patients included in the study generated 71 blastocysts, corresponding to a 69% blastocyst rate calculated per number of fertilized oocytes. This figure was similar after PB and blas-tomere biopsy. The chromosome status of the embryos that failed to develop to blastocysts was mostly aneuploid (74%). The BF was aspirated from 51 blastocysts, of which 37 were from oocytes having had PB biopsy and 14 from embryos biopsied at the cleavage stage. Analysis of the Blastocelic Fluid for the Presence of DNA 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, Fertility and Sterility® and TE cells (Supplemental Tables 1 and 2, available online at www.fertstert.org). The mean amount of DNA detected, amplified from the BF, was 900.38 ng/mL (range 876.3– 939.5 ng/mL). In the 12 BF samples with no results (23.5%), failed ampli-fication occurred, as revealed by the absence of a specific band in the agarose gel run for 5 minutes. However, when the gel was electrophoresed for a longer time, a smear appeared, suggesting possible DNA degradation in the orig-inal sample (lane 6 in Supplemental Fig. 2, available online at www.fertstert.org). The subsequent labeling and hybridiza-tion led to inconclusive results. Ten of these blastocysts derived from aneuploid oocytes (n ¼ 6) or blastomeres (n ¼ 4), whereas the remaining two were predicted to be euploid by PB (n ¼ 1) or blastomere (n ¼ 1) testing. The whole embryo could be analyzed in seven cases, and the results confirmed those obtained in the previous biopsies. Chromosomal Complement of the Blastocelic Fluid in Relation to the Data Obtained by Polar Bodies or Blastomere and Trophectoderm Cells The data from the 39 BFs with informative results were eval-uated in relation to the data obtained with the use of the chro-mosomal testing in PBs (n ¼ 30) or blastomeres (n ¼ 9; Table 1). In all, BFs reflected the ploidy condition predicted by PBs in 93.3% of cases (28/30) and by blastomeres in 100% of cases (9/9), accounting for a total ploidy concor-dance of 94.9% of cases (37/39), with two cases (5.1%) discordant. In more detail, for PBs there was full chromosome concordance in 21 samples (70% of 30), partial concordance in seven samples (23.3%), and null concordance in the re-maining two samples (6.7%). For blastomeres, full chromo-some concordance was found in eight samples (88.9% of 9) and partial concordance in one sample (11.1%). TABLE 1 Overall chromosome concordance by stage at biopsy calculated per ploidy condition (euploid vs. aneuploid) and per single chromosome. Concordance, n (%) Full Partial Null Total BFs vs. PB Embryos 21 (70) 7 (23.3) 2 (6.7) 30 Chromosomes 483/483 (100) 126/161 (78.3) 36/46 (78.3) 645/690 (93.5)a BFs vs. blastomeres Embryos 8 (88.9) 1 (11.1) 0 9 Chromosomes 192/192 (100) 11/24 (46) 203/216 (94.0) BFs vs. TE cells Embryos 32 (82) 6 (15.4) 1 (2.6) 39 Chromosomes 768/768 (100) 121/144 (84.0) 15/24 (62.5) 904/936 (96.6)a TE cells vs. PBs Embryos 21 (70) 8 (26.7) 1 (3.3) 30 Chromosomes 483/483 (100) 152/184 (82.6) 22/23 (95.7) 657/690 (95.2) TE cells vs. blastomeres Embryos 7 (77.8) 2 (22.2) 0 9 Chromosomes 168/168 (100) 38/48 (79.2) 206/216 (95.4) Note: BF ¼ blastocyst fluid; PBs ¼ polar bodies; TE ¼ trophectoderm. a P.01. Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. VOL. - NO. - / - 2014 3
  • 4. ORIGINAL ARTICLE: GENETICS Altogether, a full chromosome concordance was detected in 29 BF samples with the corresponding PBs (n ¼ 21) and blastomeres (n ¼ 8) (Supplemental Table 1). A euploid condi-tion had been predicted in six cases (three after PB testing and three after blastomere testing) and confirmed in the BF of the resulting blastocysts, including one case where all the aneuploidies detected in PB1 were compensated by reciprocal aneuploidies in PB2 (Fig. 1). The remaining 23 samples were confirmed to be aneuploid (see an example in Fig. 2). Partial concordance was detected in 8 samples, which were all predicted to be aneuploid by PBs (n ¼ 7) or blasto-mere (n ¼ 1). Here, the ploidy condition was concordant, but BFs showed aneuploidies that were only partially pre-dicted by the analysis at the previous stages (Supplemental Table 2). An example is shown in Supplemental Figure 3 (available online at www.fertstert.org). Null concordance was found in two samples, both defined as aneuploid by PB testing. Here, the ploidy condition found in the BFs was discordant with the prediction made by PBs (Supplemental Table 2). In one of these cases, chromosomal abnormalities were found only in the BF, whereas PBs and TE cells were euploid. In the other case, PBs predicted an em-bryo trisomic for chromosome 6, but the resulting blastocyst was euploid according to the analysis of BF, TE cells, and the whole embryo. When comparing the results from BFs and the corre-sponding TE cells, in 97.4% of sets (38/39) the ploidy condi-tion of BFs was confirmed in TE cells (Table 1). In more detail, there was full chromosome concordance in 32 cases (82% of 39), partial concordance in six cases (15.4%), and null concordance in one case (2.6%). The discordant case was classified as aneuploid by BF analysis and euploid by TE analysis with nine discordant chromosomes (Supplemental Table 2). Looking at single chromosomes in the whole dataset, the rate of concordance between BF and the other biopsies was 93.5% with PBs, 94% with blastomeres, and 96.6% with TE cells (96.6% vs. 93.5%; P.01; Table 1). No statistically significant difference was detected when the results from TE cell analysis were compared with the data obtained by PBs and blastomeres (Table 1). Chromosomal Complement of the Blastocyst Assessed by the Analysis of the Whole Embryo in Relation to the Data Obtained by Polar Bodies, Blastomeres, Blastocyst Fluid, and Trophectoderm Cells To add further information regarding the comparisons of the chromosome results obtained at each stage, the whole embryo was analyzed in 26 sets, 20 having had PB biopsy and six blas-tomere biopsy (Table 2; Supplemental Table 3, available online at www.fertstert.org). All of them had had BF and TE biopsies. The ploidy condition of the whole embryo was predicted in 95% of cases by PBs (19/20), in 100% of cases by blasto-meres (6/6), and in 100% of cases by both BF and TE cells (26/26; Table 2). The only discordant case was predicted as aneuploid for chromosome 6 by PBs and found to be euploid in the whole embryo as well as in the corresponding BF and TE cells (Supplemental Table 3). Full chromosome concordance was similar throughout all stages, namely, 80% with PBs (16/20), 100% with blastomeres (6/6), 80.8% with TE cells (21/26), and 84.6% with BF (22/26). Partial concordance was 15% with PBs (3/20), 19.2% with FIGURE 1 The aneuploidies in polar body 1 (PB1) were compensated by reciprocal aneuploidies in polar body 2 (PB2). As a result, the corresponding blastocyst was euploid according to analysis of the blastocelic fluid (BF) and trophectodem cells (TE). Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 4 VOL. - NO. - / - 2014
  • 5. TE cells (5/26), and 15.4% with BFs (4/26). Finally, null concordance was found with one PB case (5% of 20). Calculated per single chromosome, the grade of concor-dance was 98.9% with PBs, 100% with blastomeres, 97.9% with BFs, and 98.1% with TE cells. DISCUSSION The most relevant findings in our study were the detection of DNA in the majority of BFs and the strong prediction value of PB and blastomere testing on the blastocyst chromosomal status. In 23.5% of the investigated BFs, no informative DNA could be detected. No morphologic factors in the studied blas-tocysts could be recognized as able to predict the finding of DNA in BFs, although a common feature in the blastocysts with failed amplification was the presence of a poor-quality TE. On the other hand, this characteristic did not prevent the finding of DNA in the BFs of other cases. After attempting WGA, we observed that the samples that failed to amplify probably had highly fragmented DNA, as suggested by the smear observed in the agarose gel when it was electrophor-esed for 15 minutes (Supplemental Fig. 2). More cases are needed to see if there is a connection between these observa-tions and the absence of informative DNA in the BF. Technical problems could also have occurred, especially during the tubing of the BF, and this is a step that we are carefully revis-iting. We are also trying to evaluate if the time of biopsy could have an effect on the subsequent outcome. As a preliminary finding, we retrieved the BF twice from two blastocysts at 24-hour intervals. In one case, WGA failed in the first biopsy and was successful in the second sample; in the other case, Fertility and Sterility® both samples were successful and provided the same diagnosis. The presence of DNA in BFs, detected and successfully tested in 76.5% of the studied samples, is actually not surpris-ing. All cells surrounding the blastocele are metabolically active and secrete different substances, including several types of proteins (18, 19). It is well known that cell death normally occurs even in good-quality blastocysts, in both ICM and TE compartments. A study evaluating blastocysts originating from embryos with different levels of fragmenta-tion demonstrated higher levels of apoptosis in embryos of excellent morphology, suggesting a possible role of apoptosis in the regulation of cell number (20). Because the aspiration of the fluid was done by carefully avoiding the contamination by any cellular material, we are confident that the extracted DNA is a true reflection of the BF content. In addition, the detection of some differences in the chromosome status of BFs compared with that of TE cells supports a different origin of the two samples. Based on the above considerations, the presence of DNA in BFs could be consequent to its release from dead cells into the blastocelic cavity, with its actual quantity being possibly related to the rate of cell death. Therefore, there could be three different scenarios in the analysis of the BF: 1) The DNA in the BF is too fragmented or too scarce to result in successful genome amplification; 2) the DNA in the BF am-plifies and appears to be a reflection of the ploidy condition of the blastocyst, taking TE cells as the reference (TE biopsy being the conventional procedure for PGS of blastocysts); and 3) the DNA in the BF is discordant with the chromo-somal complement of the blastocyst, taking TE cells as the reference. FIGURE 2 A gain for chromosome 8 in PB1 and for chromosomes 15 and 22 in PB2 predicted a blastocyst monosomic for chromosomes 8, 15, and 22. This condition was confirmed in both the BF and the TE cells. Abbreviations as in Figure 1. Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. VOL. - NO. - / - 2014 5
  • 6. ORIGINAL ARTICLE: GENETICS Regarding the first scenario, this happened in our dataset at a frequency of 23.5%, with no informative DNA found in 12 of the 51 studied samples. As already mentioned, no fac-tors were identified that could be associated with the negative or positive finding of DNA. For the second scenario, the analysis of the BF was in agreement with that of the corresponding TE cells in 38 samples (74.5% of 51 samples) with full chromosome concordance per single chromosome in 32 of them. Finally, the remaining set represented the third scenario (2% of 51 samples), with BF showing multiple chromosome anomalies in a blastocyst that was predicted to be euploid by PB and TE analysis (Table 1). In summary, the ploidy condition between BFs and TE cells was found to be concordant in all cases except one (38/39, 97.4%), suggesting that BF could represent a valuable material for clinical use, provided that the proportion of sam-ples with informative DNA is raised to levels similar to those obtained by conventional biopsy procedures. Looking at single chromosomes, the global evaluation of the data in the 39 samples with successful amplification and diagnosis of DNA in the BF demonstrated full concordance between BFs and TE cells in 82% of cases (32/39), with the DNA retrieved by blastocentesis reflecting the exact chromo-somal content of TE cells. In the remaining sets, some chro-mosome variations were detected between BFs and TE cells, which is not surprising if we consider the possible origin of DNA in BF, which could also contain DNA released from the ICM (20). As presented in Table 1, the BF was extremely sensitive, similarly to TE cells, in revealing all of the chromo-some variations that occurred during embryo development. As expected, the concordance per single chromosome was significantly lower with PBs compared with TE cells, confirm-ing that the BF collects the DNA produced by TE cells as well as the ICM. In view of our results, because the artificial collapse of the blastocele is routinely applied in the vitrification of expanded and hatching blastocysts (21), it would be advis-able to store the BF and to have it at least as a back-up sample until further refinement of its use as alternative DNA source is reached. The second relevant finding coming from our study is the high level of prediction of the blastocyst chromosomal status made by the analysis done at the previous stages. Taking TE cells as the reference (Table 1), the prediction of ploidy made by PB or blastomere testing was confirmed in 97.4% of cases (96.7% and 100%, respectively). When considering the number of abnormalities per single chromosome, PBs and blastomeres had similar prediction power of the chromo-some complement of TE cells (95.2% and 95.4% respectively). A similar trend was observed when taking the whole embryo as the reference (Table 2), with the ploidy condition confirmed in 95% of PBs and 100% of blastomeres but the concordance per single chromosome rising to 98.9% and 100%, respec-tively. In other words, TE cells are only partially representa-tive of the whole blastocysts, because the ICM could have some differences regarding chromosome condition. It can be speculated that the BF collecting DNA from both TE and ICM could be more sensitive in defining the whole blastocyst chromosome status. The high prediction level of PBs of the whole embryo chromosome condition is in contrast with other authors claiming that testing at the PB stage is the less accurate method compared with blastomeres, mainly owing to the high incidence of post-zygotic events (22). In this respect, it must be mentioned that in that work the authors assumed that TE cells are representative of the blasto-cyst chromosome complement, an assumption that they corroborated in another work with the use of fluorescence in situ hybridization (23). This is probably true in most cases, especially when considering our data as well as the positive clinical outcome associated with PGS on blastocysts (10, 11, 24). However, when we analyzed the whole embryo and compared the results with those derived from TE cells, the concordance per single chromosomes was 97.9% (Table 2; Supplemental Table 3). Based on these findings, taking TE cells as the point of reference to evaluate the power of prediction of PGS made at previous stages could be not totally correct. On the other hand, the capacity of BF in TABLE 2 Overall chromosome concordance by stage at biopsy over the whole embryo calculated per ploidy condition (euploid vs. aneuploid) and per single chromosome. Concordance, n (%) Full Partial Null Total Whole embryo vs. PBs Embryos 16 (80) 3 (15) 1 (5) 20 Chromosomes 368/368 (100) 65/69 (94.2) 22/23 (95.7) 455/460 (98.9) Whole embryo vs. blastomere Embryos 6 (100) 0 0 6 Chromosomes 144/144 (100) 144/144 (100) Whole embryo vs. TE cells Embryos 21 (80.8) 5 (19.2) 0 26 Chromosomes 504/504 (100) 107/120 (89.2) 611/624 (97.9) Whole embryo vs. BF Embryos 22 (84.6) 4 (15.4) 0 26 Chromosomes 528/528 (100) 84/96 (87.5) 612/624 (98.1) Note: Abbreviations as in Table 1. Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 6 VOL. - NO. - / - 2014
  • 7. defining the whole embryo ploidy condition was 100% and 98.1% per single chromosome, suggesting that an informative BF represents a true reflection of the blastocyst chromosome condition. Blastocentesis being a less invasive method of biopsy, this strategy could become the preferred choice for PGS. We observed that the lowest rates of concordance in BFs per single chromosome were frequently related to the pres-ence of multiple abnormalities in PBs or blastomeres. It could be speculated that highly aneuploid zygotes or early embryos are predisposed to develop mosaic blastocysts, possibly owing to defective sister chromatid cohesion that can result in chromosome missegregation and aneuploidy (25). Misaligned chromosomes in a metaphase aneuploid cell or lagging chromosomes between anaphase cells also promote the formation of mosaicism. It has been reported that aneuploidy induces an increase in DNA recombination and a decrease in DNA damage repair efficiency, which are both forms of genomic instability (26). In other words, aneu-ploidy per se seems to be able to induce chromosomal insta-bility, and this could explain the type of results obtained in some cases of partial concordance (Supplemental Table 2). More on the subject is added by an experimental mouse model showing that mosaic aneuploid embryos can develop and implant in female uterine tissue and initiate the gastru-lation process, but quickly degenerate (27). Searching for the mechanism responsible for the demise of these embryos, it was found that it is caused by the activation of a spatially and temporally controlled p53-independent apoptotic mech-anism and does not result from a failure to progress through mitosis (27). Therefore, the initial state of primary aneu-ploidy resulted in a rapid evolution of mosaicism and early embryonic death. This gestational loss due to aneuploid mosaicism could account for the large proportion of human pregnancy loss before clinical recognition. The per-chromosome discordances detected in our study between BFs and TE cells, and between them and the whole embryo, could be a reflection of this mechanism which could be especially relevant in the case of oocytes carrying multiple aneuploidies (Supplemental Table 2). In conclusion, the present data confirmed the relevance of the oocyte and of the early-cleavage embryo in determining the ploidy of the resulting blastocyst. Therefore, although the incidence of post-zygotic events can not be disregarded, the oocyte and the initial stages of embryogenesis do provide reliable results for PGS (and preimplantation genetic diag-nosis). The finding of DNA in BFs, besides representing a possible alternative source of material for chromosomal anal-ysis, could contribute additional information on the study of chromosome segregation in the early embryo. REFERENCES 1. Barcroft LC,Moseley AE, Lingrel JB, Watson AJ. Deletion of the Na/K-ATPase alpha1-subunit gene (Atp1a1) does not prevent cavitation of the preimplan-tation mouse embryo. Mech Dev 2004;121:417–26. 2. Barcroft LC, Offenberg H, Thomsen P, Watson AJ. Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation. Dev Biol 2003;256:342–54. Fertility and Sterility® 3. Watson AJ, Natale DR, Barcroft LC. Molecular regulation of blastocyst for-mation. Anim Reprod Sci 2004;82:583–92. 4. Magli MC, Jones GM, Gras L, Gianaroli L, Korman I, Trounson AO. Chro-mosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in-vitro. Hum Reprod 2000;15: 1781–6. 5. Sandalinas M, Sadowy S, Alikani M, Calderon G, Cohen J, Munne S. Devel-opmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Hum Reprod 2001;16:1954–8. 6. Li M,Marin DeUgarte C, Surrey M, Danzer H, DeCherney A, Hill DL. Fluores-cence in-situ hybridization reanalysis of day-6 human blastocysts diagnosed with aneuploidy on day 3. Fertil Steril 2005;84:1395–400. 7. Hassold T, Hall H, Hunt P. The origin of human aneuploidy: where we have been, where we are going. Hum Mol Genet 2007;16:R203–8. 8. Fragouli E, Lenzi M, Ross R, Katz-Jaffe M, Schoolcraft WB, Wells D. Compre-hensive molecular cytogenetic analysis of the human blastocyst stage. Hum Reprod 2008;23:2596–608. 9. Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a random-ized pilot study. Mol Cytogenet 2012;5:2. 10. Schoolcraft WB, Katz-Jaffe MG. Comprehensive chromosome screening of trophectoderm with vitrification facilitates elective single-embryo transfer for infertile women with advanced maternal age. Fertil Steril 2013;100: 615–9. 11. Scott RT Jr, Upham KM, Forman EJ, Hong KH, Scott KL, Taylor D, et al. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil Steril 2013;100: 697–703. 12. Forman EJ, Treff NR, Stevens JM, Garnsey HM, Katz-Jaffe MG, Scott RT Jr, et al. Embryos whose polar bodies contain isolated reciprocal chromo-some aneuploidy are almost always euploid. Hum Reprod 2013;28: 502–8. 13. Fragouli E, Wells D. Aneuploidy in the human blastocyst. Cytogenet Genome Res 2011;133:149–59. 14. Palini S, Galluzzi L, de Stefani S, Bianchi M, Wells D, Magnani M, et al. Genomic DNA in human blastocoele fluid. Reprod Biomed Online 2013; 26:603–10. 15. Alpha Scientists in Reproductive MedicineESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: pro-ceedings of an expert meeting. Hum Reprod 2011;26:1270–83. 16. Magli MC, Montag M, Koester M, Muzii L, Geraedts J, Collins J, et al. Polar body array CGH for prediction of the status of the corresponding oocyte. Part II: technical aspects. Hum Rep 2011;26:3181–5. 17. Geraedts J, Montag M, Magli MC, Repping S, Handyside A, Staessen C, et al. Polar body array-CGH for prediction of the status of the corresponding oocyte. Part I: clinical results. Hum Reprod 2011;26: 3173–80. 18. d’Alessandro A, Federica G, Palini S, Bulletti C, Zolla L. A mass spectrometry-based targeted metabolomics strategy of human blastocoele fluid: a prom-ising tool in fertility research. Mol Biosyst 2012;8:953–8. 19. Jensen PL, Beck HC, Petersen J, Hreinsson J, Wanggren K, Laursen SB, et al. Proteomic analysis of human blastocoel fluid and blastocyst cells stem cells and development. Stem Cells Dev 2013;22:1126–35. 20. Hardy K, Stark J, Winston RML. Maintenance of the inner cell mass in human blastocysts from fragmented embryos. Biol Reprod 2003;68: 1165–9. 21. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitri-fication improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod 2006;21:3246–52. 22. Capalbo A, Bono S, Spizzichino L, Biricik A, Baldi M, Colamaria S, et al. Sequential comprehensive chromosome analysis on polar bodies, blasto-meres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development. Hum Reprod 2013;28:509–18. VOL. - NO. - / - 2014 7
  • 8. ORIGINAL ARTICLE: GENETICS 23. Capalbo A, Wright G, Elliott T, Ubaldi FM, Rienzi L, Nagy ZP. FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diag-nostic impact of mosaicism at the blastocyst stage. Hum Reprod 2013;28: 2298–307. 24. Forman EJ, Upham KM, Cheng M, Zhao T, Hong KH, Treff NR, et al. Compre-hensive chromosome screening alters traditional morphology-based embryo selection: a prospective study of 100 consecutive cycles of planned fresh euploid blastocyst transfer. Fertil Steril 2013;100:718–24. 25. Watrin E, Prigent C. Sister chromatid cohesion and aneuploidy. In: Storchova Z, editor. Aneuploidy in health and disease. Intech. Accessed May 16, 2012. Available from: www.intechopen.com/books/aneuploidy-in-health-and-dis ease/sister-chromatid-cohesion-and-aneuploidy. 26. Sheltzer JM, Blank HM, Pfau SJ, Tange Y, George BM, Humpton TJ, et al. Aneuploidy drives genomic instability in yeast. Science 2011;333:1026–30. 27. Lightfoot DA, Kouznetsova A, Mahdy E, Wilbertz J, H€o€og C. The fate of mosaic aneuploid embryos during mouse development. Dev Biol 2006; 289:384–94. 8 VOL. - NO. - / - 2014
  • 9. SUPPLEMENTAL FIGURE 1 Aspiration of the blastocyst fluid from an expanded blastocyst. An intracytoplasmic sperm injection pipette was inserted in the point of contact between two trophectoderm cells to minimize the amount of crossed cytoplasm. Attention was paid to avoid the aspiration of cellular material. Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. Fertility and Sterility® VOL. - NO. - / - 2014 8.e1
  • 10. ORIGINAL ARTICLE: GENETICS SUPPLEMENTAL FIGURE 2 Amplification product of seven blastocelic fluid (BF) samples on 1.5% agarose gel. (A) Gel electrophoresed for 5 minutes at 100 V, and (B) the same gel run for 15 minutes. The lane contents are labeled as follows: one kb ladder, followed by seven BF samples. The last two lanes are positive and negative control samples, respectively. The amplification band is strong in all samples and absent in sample 6 (A). When the gel was run for 15 minutes, a smear appeared in sample 6 (B). Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 8.e2 VOL. - NO. - / - 2014
  • 11. SUPPLEMENTAL FIGURE 3 Fertility and Sterility® A gain for chromosome 16 in the polar body 2 (PB2) predicted a blastocyst monosomic for chromosome 16. This condition was found in both the blastocelic fluid (BF) and trophectoderm cells (TE), but with the addition of monosomy 9. Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. VOL. - NO. - / - 2014 8.e3
  • 12. ORIGINAL ARTICLE: GENETICS SUPPLEMENTAL TABLE 1 Chromosomal status in blastocelic fluid (BF) samples showing full correspondence with the results predicted by polar bodies (PBs) or blastomeres. Sample ID Age (y) PB1 PB2 Blastomere BF TE 3 38 euploid loss 2 – gain 2 gain 2 5 38 gain 8 gain 15, 22 – loss 8,15,22 loss 8,15,22 7 36 – loss 14 loss 14 loss 14 9 36 – euploid euploid euploid 13 36 gain 4, 5, 6, 7, 9, 11, 12, 15, 19, 20, X loss 1, 2, 3, 8, 10, 13, 14, 16, 18 gain 1, 2, 3, 8, 10, 13, 14, 16, 18 loss 4, 5, 6, 7, 9, 11, 12, 15, 19, 20, X – euploid euploid 19 42 euploid euploid – euploid euploid 21 38 – – euploid euploid euploid 22 38 – – euploid euploid euploid 24 41 – – loss 16 loss 16 loss 16 28 42 euploid loss 21 – gain 21 gain 21, loss 1 29 41 euploid euploid – euploid euploid 33 35 euploid gain 15 – loss 15 loss 15 57 41 euploid gain 19, 21 loss 18, 22 – gain 18, 22 loss 19, 21 gain 18, 22 loss 19, 21 58 41 euploid loss 15 – gain 15 gain 15 85 36 loss 22 loss 14 – gain 14, 22 gain 14, 22 86 36 gain 9q loss 9p, 15 loss 9 – gain 9q, 15 gain 9q, 15 87 36 gain 16 loss 13 loss 16 – gain 13 gain 13 88 36 loss 15 gain 15 (chromosome) – loss 15 loss 15 89 36 gain 4q (chromosome) gain 16 loss 4q – loss 16, 4q loss 16, 4q 90 36 euploid gain 13 – loss 13 loss 13 111 37 – – loss 8, 16 loss 8, 16 loss 8, 16 112 37 – – gain 2, 7, 21 loss 10 gain 2, 7, 21 loss 10 gain 2, 7, 21 loss 10, 11, 16 114 37 – – gain 8, 16 gain 8, 16 gain 8, 16 116 40 gain 21 loss 13 gain 22 – gain 13 loss 21, 22 gain 13 loss 21, 22 118 40 gain 15, 21 gain 22 loss 15, 21, 22 gain 10 loss 15, 21, 22 119 40 gain 7q loss 4, 7p loss 7 – gain 4, 7p (chromosome) gain 4, 7p (chromosome) 121 40 loss 9 euploid – gain 9 gain 9 124 40 gain 18 loss 16 gain 16 loss 18, 22 – gain 22 gain 22 126 40 gain 21 loss 18 gain 18 loss 8 – gain 8 loss 21 gain 8 loss 21 Note: In all samples except three (samples 28, 112, and 118) the full concordance also included cells biopsied from the trophectoderm (TE). All gains and losses observed in PBs were due to chromatid predivision except where indicated. Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 8.e4 VOL. - NO. - / - 2014
  • 13. SUPPLEMENTAL TABLE 2 Fertility and Sterility® Chromosomal status in BF samples and corresponding TE showing partial or null correspondence with the results predicted by PBs or blastomeres. Sample ID Age (y) PB1 PB2 Blastomere BF TE 1 41 loss 16 loss 12 – gain 12,16 loss 15 gain 12,16 loss 15 4 38 euploid gain 22 – loss 17,22 loss 17,22 11 36 euploid gain 16 – loss 9, 16 loss 9, 16 6 37 gain 9 loss 1 gain 1, 16, 22 loss 9 – gain 12 loss 15, 16 loss 16,22 8 36 – – gain 5, 10, 17, 21 loss 1, 6, 14, 15, 22 gain 8, 13, 21 loss 1, 5, 17, 18, X gain 21 10 36 gain 1, 2, 4, 8, 11, 12, 13, 15, 20, 22 loss 3, 5, 9, 10, 16, 18, 19, 21, X euploid – loss 12 loss 12 18 42 gain 4, 19 – gain 5 loss 4 – gain 2, 4, 8, 12, 20 loss 5, 13, 14 gain 2,8,17,18 loss 10,13,19 70 38 gain 20 loss 21 euploid – gain 16 loss 20 gain 16 loss 20 2 38 euploid euploid – gain 5, 8, 11, 12, 15, 19 loss 3, 9, 16 euploid 117 40 loss 6, 22 gain 22 – euploid euploid Note: Partial concordance was found in eight sets. In the first three samples (samples 1, 4, and 11), BFs corresponded with the predictions made by PBs and blastomeres with the addition of new aneuploidies. In the subsequent five samples (samples 6, 8, 10, 18, and 70), only some of the anomalies predicted by PBs and blastomeres appeared in BFs, and new ones were detected. In two cases (samples 10 and 70), BFs and TE cells fully matched. Null concordance was detected in the last two samples (2 and 117), where the chromosomal status of the DNA retrieved by the BF did not match with that predicted by PBs. All gains and losses observed in PBs were due to chromatid predivision. Abbreviations as in Supplemental Table 1. Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. VOL. - NO. - / - 2014 8.e5
  • 14. SUPPLEMENTAL TABLE 3 Chromosome status in PBs or blastomeres compared with the results obtained in TE cells and the whole embryo. Sample ID Age (y) PB1 PB2 Blastomere BF TE Whole embryo 19 42 euploid euploid - euploid euploid euploid 21 38 – – euploid euploid euploid euploid 22 38 – – euploid euploid euploid euploid 24 41 – – loss 16 loss 16 loss 16 loss 16 33 35 euploid gain 15 – loss 15 loss 15 loss 15 57 41 euploid gain 19,21 loss 18,22 – gain 18,22 loss 19,21 gain 18, 22 loss 19, 21 gain 18, 22 loss 19,21 58 41 euploid loss 15 – gain 15 gain 15 gain 15 86 36 gain 9q loss 9p,15 loss 9 – gain 9q,15 gain 9q, 15 gain 9q, 15 87 36 gain 16 loss 13 loss 16 – gain 13 gain 13 gain 13 88 36 loss 15 gain 15 (chromosome) – loss 15 loss 15 loss 15 89 36 gain 4q (chromosome) gain 16 loss 4q – loss 16,4q loss 16, 4q loss 16, 4q 90 36 euploid gain 13 – loss 13 loss 13 loss 13 111 37 – – loss 8, 16 loss 8, 16 loss 8, 16 loss 8, 16 114 37 – – gain 8, 16 gain 8, 16 gain 8, 16 gain 8, 16 116 40 gain 21 loss 13 gain 22 – gain 13 loss 21, 22 gain 13 loss 21, 22 gain 13 loss 21, 22 119 40 gain 7q loss 4, 7p loss 7 – gain 4, 7p (chromosome) gain 4, 7p (chromosome) gain 4, 7p (chromosome) 121 40 loss 9 euploid – gain gain 9 gain 9 124 40 gain 18 loss 16 gain 16 loss 18, 22 – gain 22 gain 22 gain 22 126 40 gain 21 loss 18 gain 18 loss 8 – gain 8 loss 21 gain 8 loss 21 gain 8 loss 21 18 42 gain 4, 19 gain 5 loss 4 – gain 2, 4, 8, 12, 20 loss 5, 13, 14 gain 2, 8, 17, 18 loss 10, 13, 19 loss 5, 19 28 42 euploid loss 21 – gain 21 gain 21 loss 1 gain 21 70 38 gain 20 loss 21 euploid – gain 16 loss 20 gain 16 loss 20 gain 7 loss 20 85 36 loss 22 loss 14 – gain 14, 22 gain 14, 22 gain 4, 14, 22 112 37 – – gain 2, 7, 21 loss 10 gain 2, 7, 21 loss 10 gain 2, 7, 21 loss 10, 11, 16 gain 2, 7, 21 loss 10 117 40 loss 6 (chromosome), 22 gain 22 – euploid euploid euploid 118 40 gain 15,21 gain 22 – loss 15, 21, 22 gain 10 loss 15,21,22 gain 10 loss 15,21,22 Note: The samples 19 to 126 are those with full concordance throughout all the studied stages. All gains and losses observed in PBs were due to chromatid predivision except where indicated. Abbreviations as in Supplemental Table 1. Gianaroli. Blastocentesis as a source of DNA. Fertil Steril 2014. 8.e6 VOL. - NO. - / - 2014 ORIGINAL ARTICLE: GENETICS