Asian Pacific Journal of Reproduction 2014; 3(2): 85-89 85

Asian Pacific Journal of Reproduction

Journal homepage: www.apjr.net

Document heading doi: 10.1016/S2305-0500(14)60009-9

C orrelation between embryo morphology and development and

chromosomal complement
Vy Phan*, Eva Littman, Dee Harris, Antoine La
Red Rock Fertility Center, 6420 Medical Center ST, STE 100, Las Vegas, Nevada, USA

ARTICLE INFO ABSTRACT

Article history: Objective: To analyze the correlation between embryo morphology and the chromosomal status
Received 21 February 2014
Received in revised form 25 February 2014
using the array comparative genomic hybridization [array comparative genomic hybridization
Accepted 25 February 2014 (a-CGH)] technique for screening 23 chromosome pairs in a single blastomere biopsy from Day
Available online 20 June 2014 3 embryos. Methods: One thousand five hundred and fifty seven embryos were included from
203 cycle ICSI patients undergoing preimplantation genetic screening. The 23 chromosome pairs
Keywords: were analyzed by blastomere biopsy from day 3 embryos using a-CGH array method. Embryo
Aneuploidy development rate, fragmentation rate and chromosome status of the analyzed blastomeres
Blastomere biopsy were recorded and correlated with the aCGH results. Results: The incidence of chromosomal
Cleavage rate abnormalities was significantly higher in slow-and fast cleaving embryos at day 3 after
Chromosomal abnormalities
insemination. The incidence of fragmentation and the type of fragmentation was associated with
Embryo fragmentation
Embryo morphology an increased incidence of chromosomal abnormalities. The symmetry of the blastomeres also
CGH correlated with the aneuploidy rates. Conclusions: Embryo development rate and morphological
parameter such as degree, type of fragmentation and the symmetry of the blastomeres to a large
extent reflect the cytogenetic status of the embryo and thus are important in the selection of
embryos with the highest implantation potential.

5-7 chromosomes, previous studies showed that embryos

1. Introduction containing unevenly sized blastomeres have an increased
One of the key factors for a successful outcome in an
aneuploidy rate[9,10]. Further, there is evidence that growth
assisted reproductive program is the selection of embryos retardation in addition to accelerated cleavage could
with the highest developmental potential. F or many be an indication of chromosomal abnormalities [11-13].
Other studies have demonstrated increased chromosomal
years embryos have been selected based on parameters
considered important quality indicators, such as abnormality rates with increased degree of fragmentation or
fragmentation, cell number and cell size[1-7]. poor embryo morphology[11-13].
M ost studies published have been based on the
The development and implementation of techniques for
preimplantation genetic diagnostic programs have made technique of Fluorescence In Situ Hybridization (FISH)
it possible to assess the chromosomal constitution without results. This form of evaluation consists of testing 5 to 12
destroying the embryo. It has been suggested that pronuclear chromosomes from either single or dual cell biopsy in an
morphology could be indicative of embryo quality[1,8]. Using attempt to predict the chromosomal status of the whole
FISH technique for pre-implantation genetic screen for
embryo [10,12,13]. Therefore, the information obtained is not
always representative of the real chromosomal status. The
adaptation of a-CGH to single cells has allowed the study of
*Corresponding author: Vy Phan, Red Rock Fertility Center, 6420 Medical Center ST, the full karyotype of blastomeres, thus identifying the true
STE 100, Las Vegas, Nevada, USA
Tel: (1) 702 262 0079 level of aneuploidy in cleavage-stage embryos, which was
Fax: (1) 702 685 6910;
E-mail: [email protected] then reported to affect 75% of them[14-17].
86 Vy Phan et al./ Asian Pacific Journal of Reproduction (2014)85-89

The aim of this study was to analyze the correlation number; degree of fragmentation (without fragmentation,
between embryo morphology and the chromosomal status less than 5%, 6% to 15 %; 16% to 30% and more than 30%
using the a-CGH technique for screening 23 chromosome fragmentation); localization of fragments (local or dispersed);
pairs in a single blastomere biopsy from Day 3 embryos. equally or unequally sized blastomeres).
Biopsy procedures were carried out on day 3 (66-68 hours
after insemination). One blastomere was gently aspirated
2. Materials and methods with the use of a biopsy pipette. After blastomere biopsy,
embryos were thoroughly rinsed and transferred to a new
2.1. Patient population, embryo culture and biopsy dish of Global media with 10% SSS and cultured to day 5 and
day 6. The biopsied blastomere was transferred to the tubes
203 patients undergoing in vitro fertilization (IVF) treatment and sent to a Genetics laboratory for chromosome evaluation
and preimplantation genetic screening (PGS) with aCGH at by a-CGH. Embryos marked as euploid were chosen for
Red Rock Fertility Center were included in this study. The transfer or frozen.
study was conducted after obtaining the Institutional Review
Board’s approval. The average maternal age of patients was 2.3. Statistical analysis
34.7 years (range 29-41 years). Patients underwent one of the
following orarian stimulation protocols; luteal phase Lupron Data was analyzed by chi-square analysis and relative risk
suppression (Leuprolide acetate; TAP Pharmacceuticals, test (RR test).
L ake F orest, IL ) with or without oral contraceptive
pretreatment; gonadotropin-releasing hormone (GnRH)
antagonist prevention of premature ovulation with cetrorelix 3. Results
( C etrotide; EMD D erono, R ockland, MA ) or ganirelix
(Organon USA, Roseland NJ). In the antagonist protocol, the 1 257 cleavage-stage embryos were biopsied and analyzed
GnRH antagonist was added when a lead follicles measured for aneuploid. A total of 783/1257 (62.3%) were aneuploidy
≥14 mm. Controlled ovarian hyperstimulation was performed and 474 / 1257 ( 37 . 7 %) were euploid. A ll embryos were
with human menopausal gonadotropin (Menopur; Ferring observed until the end of day 6 for developmental progress.
Pharmaceuticals, Parsippany, NJ), recombinant luteinizing 572 blastocysts developed from biopsied embryos, of which
hormone (LH, Luveris, EMD Serono), and/or recombinant 257 (32.8%) were developed from aneuploidy embryos and
FSH ( F ollistim, O rganon USA ; G onal- F , EMD S erono ) . 315 (66.5%) were developed from euploidy embryos. The
Cycles were monitored with serum estradiol levels and competence to develop to blastocyst stage was decrease 2
transvaginal ultrasounds. When at least 2 follicles measured times in aneuploidy embryos compare to euploidy embryos
≥18 mm, 5 000-10 000 units of urinary hCG (Novarel; Ferring (32.8% vs. 66.5%); RR = 2; P<0.001 (Table 1).

P harmaceuticals ) were administered subcutaneously.

Ultasound-guided oocyte retrieval was performed 36 hours Table 1
Development of biopsied embryos to blastocyst stage.
after hCG administration.
Development of D3 embryos Aneuploid Euploid RR
All mature oocytes were fertilized by intra-cytoplamic Slow/ Arrested 526(67.2%) 159(33.5%)
a
1
sperm injection ( ICSI ) method. E mbryos were cultured Blastocysts 257(32.8%) 315(66.5%)
b
2
using Global media (LifeGlobal) with 10% Serum Substitute Total 783 474 -
Supllement (SSS) (Irvine Scientific) under triple gas incubator a vs. b p <0.001
(6.5% CO2; 5% O2 and 88.5% N2).
A total of 1 257 embryos were biopsied on day 3 of embryo F igure 1 shows the results of 1 257 embryos which
development and underwent aneuploidy screening with underwent tested for aneuploidy. The results were analyzed
aCGH. After biopsied, the embryos were culture until day for each cellular stage in details, the lowest incidence of
5 or day 6 of development. Euploid embryos were either chromosomal abnormalities was found in embryo with 8 cells
tranferred to the uterus or frozen for future use. (53.9%) and the highest was found in embryos with 4-5 cells
(87.2%). In human in-vitro fertilization, embryos usually
2.2. Embryo scoring develop to 8 cell stage at 54-72 hours after fertilization. In
our study, we assessed the embryos at 66-68 hours after
Oocytes were checked for the presence of pronuclei and
fertilization. Our results show that slow developed embryos
polar bodies 16-18 hours after ICSI. Fertilized oocytes were
that have 4-6 cells have significantly higher aneuploidy rate
cultured and scored 66-68 hours after insemination for: cell
of 83.1%, nearly 1.5 times higher than embryos which have
 Vy Phan et al./ Asian Pacific Journal of Reproduction (2014)85-89
87

7-9 cells (RR= 83.1/56.3= 1.476; P<0.001) and nearly 1.3 As shown in Table 3, aneuploidy rate was related to the
times higher than embryo which have more than 9 cells (RR= location of fragmentation. Aneuploidy rate was higher 1.9
83.1/65.7= 1.265; P<0.001). Embryos with fast development times in embryos with fragment located scattered compare
also have aneuploidy rate nearly 1.2 times higher than with embryo with fragment concentrated in the peripheral
embryos which have 7-9 embryos (RR= 65.7/56.3= 1.167; area (RR=77.6/40.1=1.94; P<0.001).
P<0.001). Table 4 shows that embryos with uneven blastomeres have
1.8 times higher aneuploidy rate compared to embryos with
even blastomeres (RR=81.6/44.1=1.85; P<0.001).
Aneuploidy rates and cellular stage,
66-68 hours after insemination
Table 3
4-5 cells 87.20%
6 cells
Aneuploidy rate and the location of fragment.
80%
7 cells 66.10%
#
tested for
Location Aneuploid Euploid RR
8 cells 53.90% aneuploid
9 cells 60.30% % fragmentation 28 8(28.6%) 20(71.4%) -
10 cells 64.60%
C oncentrated in 191(40.1%) 285(59.9%)
a
476 1
＞10 cells 68.10%
the peripheral
Scattered 584(77.6%) 169(22.4%) 1.94
b
753
Figure 1. Chromosome abnormalities and cellular stage. Total 1257 783 474 -

The numerical analysis of chromosomes was increased a vs. b P<0.001.

with the the percentage of fragmentation. T able 2
Table 4
shows that embryos with a lot of fragmentation 16 % -
Aneuploidy rate and the symmetry of the blastomeres.
30% have the highest aneuploidy rate (75.1%), 1.14 times Symmetry of the
#
tested for
Aneuploid Euploid RR
higher than embryos with moderate fragmentation blastomeres aneuploid
6 % - 15 % ( RR= 75 . 1 / 65 . 7 = 1 . 14 , P< 0 . 025 ) ; 1 . 35 times Even blastomeres 286(44.1%) 362(55.9%)
a
648 1

higher than embryos with little fragmentation 1 % - 5 % Uneven blastomeres 609 497(81.6%)
b
112(18.4%) 1.85
(RR=75.1/55.4=1.35; P<0.001) and 2.6 times higher than Total 1257 783 474 -
embryos without fragmentation ( RR= 75 . 1 / 28 . 6 = 2 . 63 ; a vs. b P<0.001
P<0.001). The aneuploidy rate in embryos with moderate
fragmentation was nearly 1 . 2 times higher than in
embryos with little fragmentation ( RR= 65 . 7 / 55 . 4 = 1 . 18 ; 4. Discussion
P<0.005) and 2.3 times higher than in embryos without
The effectiveness of chromosomal screening methods
fragmentation ( RR= 65 . 7 / 28 . 6 = 2 . 29 ; P< 0 . 001 ) . T he
depends on the ability to accurately distinguish euploid
aneuploidy rate in embryo with little fragmentation was
embryos from those affected by aneuploidy. Almost all
higher 1.9 times compared to embryo without fragmentation
previous pre-implantation genetic screening (PGS) studies
(RR=55.4/28.6=1.94; P<0.001) (Table 2).
have been based upon the use of fluorescence in situ
hybridization (FISH). Although FISH has allowed accurate
Table 2
screening of restricted numbers of chromosomes, the method
Chromosomal abnormalities and percentage of
fragmentation. is limited in that less than one-half of the chromosomes
#tested for can be enumerated in each biopsied cell. T he use of
Fragmentation Aneuploid Euploid RR
aneuploid a-CGH allows all of the chromosomes to be evaluated,
8(28.6%) 20(71.4%)
a
0% 28 1 thus revealing nearly 100% of aneuploid embryos[14,15].
1%-5% 306(55.4%) 246(44.6%) 1.94
b
552
Additionally, the a-CGH method provides the advantage of
6%-15% 276(65.7%) 144(34.3%) 2.29
c
420
avoiding the technically challenging process of cell fixation
16%-30% 193(75.1%) 64(24.9%) 2.63
d
257
Total 1257 783 474 - on a microscope slide. The data from this study indicated
a vs. b P< 0.001; b vs. c P<0.005; c vs. d P < 0.05 that selected morphology features and embryo development
rate were related to the chromosomal status of the embryo.
88 Vy Phan et al./ Asian Pacific Journal of Reproduction (2014)85-89

It has been suggested that good embryos should cleave demonstrates that the embryo development rate and
at an optimal cleavage rate[7,18-20]. Embryos which cleave morphological parameters such as degree, type of
either too fast or too slow usually indicate a compromised fragmentation, asymmetry of the blastomeres to a large
developmental potential. In this study, embryos with a slow extent reflect the cytogenetic status of the embryo and thus
cleavage rate resulting in <7 cells and embryos with fast are important in the selection of embryos with the highest
cleavage rate (>9 cells) at 68 h after fertilization showed an implantation potential. There is still an urgent need to
increased chromosomal abnormality rate from 1.5-1.2 times. clarify how normal an embryo needs to be in order to be able
In this study, we found that 62.3% of cleavage embryos to implant and give rise to a healthy baby. We do not know
were aneuploid. 66.5% of euploid embryos on day 3 were to what extent chromosomal abnormalities compromise the
capable to develop to the blastocyst stage whereas only developmental potential of the embryo and what, if any
32.8% of aneuploid day 3 embryo progressed to blastocyst. corrective mechanisms exist within the embryo that may
It is accordance with previous suggestion that culturing compensate for various degrees of chromosomal errors.
human embryos to the blastocyst stage instead of cleavage
stage will enable the selection and identification of healthy,
chromosomally normal embryos endowed with high potential Conflict of interest statement
for implantation[21,22]. This study helps to further clarify this
well-known observation. We declare that we have no conflict of interest.
In our study uneven blastomeres were associated with
high incidence of aneuploidy nearly 1.8 times that was
accordance with previous conclusion from FISH studies that References
blastomere asymmetry has been linked to reduced embryo
competence, reduced the implantation rate [9,10]. [1] Alpha Scientists in Reproductive Medicine and ESHRE Special
Finally, increasing amounts of fragmentation in the embryos Interest Group of Embryology. The Istanbul consensus workshop

at 68 h after fertilization was significantly correlated with on embryo assessment: proceedings of an expert meeting. Reprod

increased chromosomal abnormality rates. This finding is in Biomed Online 2011; 22: 632-646.

accordance with previous publications[10,11,13]. Assuming that [2] Montag M, Liebenthron J, Koster M. Which morphological scoring

an increased chromosomal abnormality rate is associated system is relevant in human embryo development? Placenta 2011;

with a decreased implantation and pregnancy potential, this 32: S252-S256.

[3] Racowsky C, Ohno-Machado L, Kim J, Biggers JD. Is there an
could explain the lowered implantation and pregnancy rates
advantage in scoring early embryos on more than one days? Hum
after transfer of fragmented embryos as found in several
Reprod 2009; 24: 2104-2113.
studies[23,24]. Ebner et al. found an increased malformation
[4] Racowsky C, Vernon M, Mayer J, Ball GD, Behr B, Pomeroy KO,
rate after transfer of highly fragmented embryos and the
et al. Standardization of grading embryo morphology. Fertil Steril
authors concluded that this might be due to a higher
2010; 94: 1152-1153.
percentage of chromosomal disorders. In the present of
[5] Racowsky C, Stern JE, Gibbons WE, Behr B, Pomeroy KO, Biggers
scattered fragmentation, the occurrence of chromosomal
JD. National collection of embryo morphology data into Society
abnormalities is significantly higher compared to when
for Assisted Reproductive Technology Clinic Outcomes Reporting
fragments are concentrated in one area. When fragmentation
System: associations among day 3 cell number, fragmentation and
was scattered, it will affect the cell-to-cell contacts,
blastomere asymmetry, and live birth rate. Fertil Steril 2011; 95:
compaction and blastocyst formation[25].
1985-1989.
In conclusion, we found a high incidence of chromosomal
[6] VanRoyen E, Mangelschots K, De Neubourg D, Laureys I, Rychaert
abnormality in embryos from couples participating in
G, Gerris J. Calculating the implantation potential of day 3 embryos
an assisted reproductive program. F urther, this study
in women younger than 38 years of age: a new model. Hum Reprod
 Vy Phan et al./ Asian Pacific Journal of Reproduction (2014)85-89
89
2001; 16: 326-332. genomic hybridization. Hum Genet 2000; 106: 210-217.
[7] Vernon M, Stern JE, Ball GD, Wininger D, Mayer J, Racowsky C. [17]Well D, Delhanty JDA. Comprehensive chromosomal analysis of
Utility of the national embryo morphology data collection by the human preimplantation embryos using whole genome amplification
Society for Assisted Reproductive Technologies (SART): correlation and single cell comparative genomic hybridization. Mol Hum
between day 3 morphology grade and live birth outcome. Fertil Reprod 2000; 6: 1055-1062.
Steril 2011; 95: 2761-2763. [18]Cruz M, Gadea B, Garrido N, Pedersen KS, Martinez M, Perez-Cano
[8] Zollner U, Zollner KP, Steck T, Dietl J. Pronuclear scoring: time for I, et al. Embryo quality, blastocyst and ongoing pregnancy rates in

the international standardization. J Reprod Med 2003; 48: 365-369. oocyte donation patietns whose embryos were monitored by time-
[9] Hardarson T, Hanson C, Sjogren A, Lundin K. Human preembryos lapse imaging. J Assited Reprod Genet 2011; 28: 569-573.
with unevenly sized blastomeres have lower pregnancy and [19]Hashimoto S, Kato N, Saeki M, Morimoto Y. Selection of high
implantation rates: indication for aneuploidy and multinucleation. potential embryos by culture in poly (dimethylsiloxane) microwells
Hum Reprod 2001; 16: 313-318. and time lapse imaging. Fertil Steril 2012; 97: 332-337.
[10]Munne S. Chromosome abnormalities and their relationship to [20]Messeguer M, Herrero J, Tejera A, Hilliasoe KM, Ramsing NB,
morphology and development of human embryos. Reprod Biomed Remohi J. The use of morphokinetics as a predictor of embryo

Online 2006; 12: 234-253. implantation. Hum Reprod 2011; 26: 2658-2671.
[11]Bialanska M, Tan SL, Ao A. Chromosomal mosaicism throughout [21]Eaton J, Hacker M, Harris D, Thornton K, Penzias A. Assessment
human preimplantation development in vitro: incidence, type, and of day 3 morphology and euploidy for individual chromosomes in
relevance to embryo outcome. Hum Reprod 2002; 17: 413-419. embryos that develop to the blastocyst stage. Fertil Steril 2009; 91:
[12]Magli MC, Gianaroli L, Munne S, Ferraretti AP. Incidence of 2432-2436.

chromosomal abnormalities from a morphologically normal cohort [22]Fragouli E, Lenzi M, Ross R, Katz-Jaffe M, Schoolcraft WB, Wells
of embryos in poor prognosis patients. J Assis Reprod Genet 1998; D. Comprehensive molecular cytogenetic analysis of the human

15: 297-301. blastocyst stage. Hum Reprod 2008; 23: 2596-2608.

[13]Magli C, Giannaroli L, Ferraretti AP, Lappi M, Ruberti A, Farfalli [23]Racowsky C, Combelles C, Nureddin A, Pan Y, Finn A, Miles L, et
V. Embryo morphology and development are dependent on the al. Day 3 and day 5 morphological predictors of embryo viability.
chromosomal complement. Fetil Steril 2007; 87: 534-541. Reprod Biomed Online 2003; 6: 323-331.
[14]Munne S. Pre-implantation genetic diagnosis for aneuploidy and [24]Ziebe S, Lundin K, Janssens R, Helmgaard L, Arce JC. MERIT
translocations using array comparative genomic hybridization. (Menotrophin vs. Recombinant FSH in vitro fertilization trial).
Curr Genomics 2012; 13(6): 463-470. Influence of ovarian stimulation with HP-hMG or recombinant FSH

[15]Traversa M, Marshall J, McArthur S, Leigh D. The genetic screening on embryo quality parameters in patients undergoing IVF. Hum
of preimplantation embryos by comparative genomic hybridization. Reprod 2007; 22: 2404-2413.
Repd Biol 2011; 11 (suppl. 3): 51-60. [25]Ebner T, Yaman C, Moser M, Sommergruber M, Polz W, Tews Q.
[16]V oullaire L , S later H , W illiamson R , W ilton L . C hromosome Embryo fragmentation in vitro and its impact on treatment and

analysis of blastomeres from human embryos by using comparative pregnancy outcome. Fertil Steril 2001; 76: 281-285.