Embryo morphology may change after biopsy for preimplantation genetic diagnosis

JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015

EMBRYO MORPHOLOGY MAY CHANGE AFTER BIOPSY FOR
PREIMPLANTATION GENETIC DIAGNOSIS?
Vu Van Tam* et al
SUMMARY
There are many methods for treating infertility patients, IVF plays an important role in the
field of assisted reproduction and development are increasingly widespread in the world.
However, the success rate in assisted reproduction only reached 30 - 40%. Currently, most are
choosing embryos based on morphological criteria of the embryo. However, the morphological
assessment not fully reflect the true quality of the embryos, if only based on morphological
parameters, the results of in vitro fertilization treatment is limited. To improve the quality and
refinement of some genetic diseases, preimplantation genetic diagnosis (PGD) is one of the
critical requirements, urgent and practical. On that basis, we conducted this study to evaluate
embryo morphological changes and assess some factors affecting the ability of embryo survival
and create blastocyst after biopsy. Subjects and methods: 102 surplus embryos, biopsy on day
3. Quality evaluated on three main criteria: number of blastomeres in an embryo, cytoplasm
fragments and the embryo volume. Results: After 24 hours after biopsy, survival rate of
embryos, further development of the 3 groups is 93 embryos in total 102 embryos, reached
91.17% and after 48 hours blastocyst formation is 83, reaching 81.37%. Number of embryos
that survived continued development (AA and AB) account for 65.68% of 67 embryos.
Cytoplasm fragment ratio, ZP thickness, embryo diameter between the groups did not change
significantly. Conclusion: Laser biopsy does not alter embryo morphology, viability and blastocyst
forming. The more higher maternal age, duration of infertility, FSH concentrations the less
survival rate of embryos and less forming blastocyst. IVF or ICSI did not affect the survival rate
of embryos, further development of blastocyst after embryo biopsy.
* Key words: Preimplantation genetic diagnosis; Embryo morphology; Biopsy.

BACKGROUND
Infertility is a problem that is growing
interest in the world in general and

Vietnam in particular, according to the
World Health Organization, the rate of
infertility accounts for about 10% of couples.
In Vietnam by Nguyen Viet Tien et al
(2013) [3], the rate of infertility in the
community accounts for 7.5%. Today, there
are many treatment methods for infertility

patients, in vitro fertilization plays an important
role in the field of assisted reproduction
and development are increasingly widespread
in the world. However, the success rate in
assisted reproduction only reached 30 40%, in this area there are many issues
that need to be further studied. Currently,
most are choosing embryos based on
morphological criteria of the embryo.
However, the morphological assessment

* Haiphong IVF Centre
Corresponding author: Vu Van Tam ()

26


JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015

do not fully reflect the true quality of the
embryos, if only based on morphological
parameters, the results of in vitro fertilization
treatment is limited. To improve the quality

of treatment as well as ensuring that a
generation of healthy physical, spiritual
insight, as well as screening genetic diseases,
genetic diagnosis prior to embryo transfer
(Preimplantation Genetic Diagnosis/PGD)
is one of the critical requirements, urgent
and practical. Principles of PGD technique
based on the performance of in vitro
fertilization (IVF) to create embryos, embryo
biopsy and then analyzed by chromosome
or DNA FISH technique, CGH or PCR [1].
Embryo biopsy technique is invasive
techniques, many operations require
meticulous and accurate. So in the
process of implementation may affect the
quality of the embryos. In parallel with the
work completed embryo biopsy procedure,
we evaluated embryo morphology, and
survey a number of factors that can affect
the development of embryo after embryo
biopsy. On that basis, we conducted a
study with project as "Study on some
factors affecting embryo morphology after
biopsy for genetic diagnosis before embryo
transfer" to assess the changes of embryo
morphology and some related factors can
affect the quality of the embryos after biopsy.
OVERVIEW
Genetic diagnosis before embryo transfer
(Preimplantation Genetic Diagnosis/PGD)

has been adopted in many other advanced
countries in the world from the last decade.

Principles of PGD technique based on the
performance of in vitro fertilization (IVF) to
create embryos, embryo biopsy and then
analyzed by chromosome or DNA FISH
technique, CGH or PCR (polymerase chain
reaction). General technique of PGD
composed 2 main steps: embryo biopsy
and genetic diagnosis. The findings of
several authors [1] found no difference in
success rates as well as the ability to
develop mental and physiological comparison
with young children after PGD and ICSI
infants after birth from natural conception.
Embryo biopsy was carried out in three
steps: preparation, opened the window on
the zona pellucida and took cells. There
are three main methods used to open
window on the zona pellucida: mechanical
methods, chemical or laser. Currently,
using a laser to open the zona membrane
is considered the simplest method, safe
and easy to use. The development of
embryos after biopsy depends on many
factors, the following are some relevant
factors can affect embryo morphology
after biopsy.
In 1995, using FISH, Munne found that

the percentage of embryos with diploid
and the number of fragments increases
with maternal age: 16% of women aged
20 to 34, 37% of women aged 35 to 39
and the highest 53% in women over 40
years old. This author in 2002 study
based on 1 cell (94 embryos) and the 2
cells (304 embryos) found that the rate of
diploid, fragments increases with maternal
age, from 12.2% in the age group under
35, to 31% at the age of 40 [4].
27


JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015

Studies comparing methods of in vitro
fertilization (IVF) and intracytoplasmic
sperm injection into oocytes (ICSI) that
forms of embryos from the above 2
methods are the same. But Walker. MC,
Murphy KE, Pan S (2004) showed that
the rate of chromosomal abnormalities
significantly higher in the patient group using
intracytoplasmic sperm injection, as these
patients are carriers of chromosomal
disorders, oligospermatozoa and OAT. In
this case, before making the diagnosis
should be made when testing the blood and
semen found that these patients tend to

transmit the disease to chromosome disorder.
Munne et al published results comparing
the relationship between embryo morphology
and FSH concentrations from which to
assess the association between FSH levels
with changes of embryo morphology and
genetic deviation during meiotic division
and early embryonic development. For
women under 40, high FSH levels, with
the rate of chromosomal disorders, embryos
in degree III and IV increased significantly
(p < 0.02), but not dependent on the
concentration of FSH. Rate of chromosomal
abnormalities, embryos in degree III and
IV in women under 40 have FSH levels
above 10 mIU/mL is equivalent to the rate
of chromosomal disorder in women over
age of 40 have the same concentration of
FSH. Such increase chromosomal
disorders, embryo morphology in women
over age 40 do not depend on the
concentration of FSH, which increases
the concentration of FSH associated with
reduced ovarian reserve capacity and in
accordance with rate low pregnancy in
this group of patients [9].
28

SUBJECTS AND METHODS
1. Subjects.

- Includes 102 surplus embryos of in
vitro fertilization.
The study subjects were divided into 3
groups:
- Group I: 33 embryos of patients with
age ≤ 30.
- Group II: 39 embryos of patients with
with age > 30 years old.
- Group II: 30 embryos of patients with
age > 35 years old.
* Selection criteria for embryo biopsy:
The embryos were cultured until day 3
of IVF culture medium. The uniform cell
embryos, the percentage of cytoplasmic
fragments (fragment) of less than 20%
(grade III embryos, grade IV).
* Exclusion criteria:
The early embryo grade I, grade II:
uneven cells, cytoplasmic fragments ratio
of 20% or more.
2. Methods.
* Evaluation process of fertilization and
embryo culture:
Approximately 16 - 18 hours after the
injection of sperm into the egg, the egg
was assessed whether or not fertilization.
If fertilized embryos form will appear 2PN
and 2 polary bodies. Then the embryos
were scored at each assessment time
40 hours, 68 hours and 112 hours after

fertilization and embryo morphology
evaluation after biopsy 24h, 48h. The
number of blastomere and form, number
fragments thickness of ZP, and diameter
embryos were collected to assess embryo
quality [5].


JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015

* The criteria of evaluation of embryo
quality:
Assessing the quality of day 3 embryos
cultured according to T. Ebner (2003),
and is being applied in our Center.
According to this way of evaluating the
quality embryos on day 3 of culture is
based on three main criteria: the number
of blastomere in an embryo, the embryo
evenly between embryos and the ratio
(%) between cytoplasmic fragments and
the volume of the embryo [7]:
- Number of embryos (denoted by a
number).
- Compare uniformity between blastomere
(denoted by a capital letter):
+ A: The even blastomere.
+ B: The uneven blastomere.
- Percentage of cytoplasmic fragments
(denoted by a number, is the ratio of

cytoplasmic fragments compared to the
volume of the embryo).
+ There is no cytoplasmic fragments.
+ Percentage of cytoplasmic fragments
< 20%.
+ Percentage of cytoplasmic fragments
from 20 - 50%.
+ Percentage of cytoplasmic fragments
> 50%.
- Based on the quality of embryos
according to 4 degrees is applied to
evaluate the quality of embryos at Vietnam
Military Medical University, author Andres
Salumets, 2001 [10]:
+ Grade IV: The embryonic blastomeres
of even, and no or very little (negligible)
cytoplasmic fragments.

+ Grade III: The embryonic blastomeres
of uneven and/or debris cytoplasmic ratio
< 20% of the embryo volume.
+ Grade II: The embryo fragments
cytoplasmic ratio from 20% to < 50% of
the embryo volume.
+ Grade I: The embryo fragments
cytoplasmic ratio ≥ 50% of the embryo
volume.
RESULTS
1. Some characteristics of the study
subjects.

Research was carried out on 102
patients, each patient 1 embryo is put into
research subjects, subjects were divided
into 3 groups as follows:
* The number of infertile patients of the
study group:
Of the 102 patients, we divided into
3 groups: Group I consists of 33 patients
under the age of 30 accounted for 32.35%,
group II included 39 patients aged 30 - 35
accounted for 38.24% and group III included
30 patients aged over 35 accounted for
29.41%, with the number of targets to
ensure research.
* Age and infertility duration of the study
group patients:
The average age of all study patients
was 29.3 ± 3.6 (20 to 42 years old), the
average age of patients in group I was
21.5 ± 3.2 (minimum age is 20, the highest
age is 29). Patients in group II with an
average age of 32.5 ± 2.2 (minimum age
is 30, oldest is 35). Patients in group III
with an average age of 38.4 ± 3.0 (minimum
age is 36, oldest is 42). Average duration
29


JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015

of infertility patients in group I was 3.3 ±
2.3 years, the shortest period of 1 year
and a maximum of 5 years. The patients
in group II with an average duration of
infertility was 4.1 ± 2.6 years, the shortest
time is 1 year and the longest is 7 years.
The patients in group III with an average
duration of infertility was 4.8 ± 3.4 in the
shortest time is 1 year and the longest is
12 years. Over time we see figures of the
infertile group I was the shortest duration
and infertility group III is the longest.

7.2 micron thinnest group II. However,
the difference in the number of embryos,
the rate of cytoplasmic fragments, ZP
thickness, diameter 3 embryos between
3 groups are not statistically significant.
After embryo biopsy continues to be cultured
in Cook Incubator, after that we measured
indicator such as: diameter, ZP thickness,
and the rate of blastocyst formation
respectively 24, 36h and 48h in each age
group to assess the development of embryos
after biopsy.

2. The morphological changes of
embryos after biopsy.

* Embryo survival rate, continues to

grow and the rate of blastocyst formation
after biopsy:

* Embryo morphology at 3 days before
biopsy:
Before biopsies we measured indicators
as a basis for comparison and evaluation
of morphological changes of the embryo
before and after biopsy. The evaluation
criteria were: number of blastomeres,
cytoplasmic fragments ratio, ZP thickness
and diameter of embryo. Average number of
embryos of group I, II and III, respectively,
7.8 ± 1.21, 7.9 ± 3.13, 8.2 ± 2.25; median
cytoplasmic cytoplasmic ratio respectively was
6.21 ± 1.23, 5.63 ± 1.31, 6.82 ± 1.27. Pellucida
thickness was measured by cross-sectional
profiles at random two locations of membrane,
the zona pellucida thickness average of
3 groups I, II and III, respectively, 15.70 ±
3.14, 16.20 ± 2.22, 16.60 ± 2.42. Diameter
of embryo of 3 groups corresponding
average is 151.50 ± 6.32 µm; 151.30 ±
4.92 µm; 152.50 ± 5.32 µm. Cytoplasmic
fragment ratio high as 15% in most
distributed over 35 age group and at least
in the age group under 30. The thickness of
ZP is the largest of the group III 24.4 micron,
30

After a 24-hour biopsy survival rate of
embryos, further development of the 3
groups is 93 embryos in total 102 embryos,
achieved 91.17% and the group I, II and
III was 93.93%; 92%, 31%, 86.66%, and
by day 5 blastocyst formation rate of the
whole group was 83 embryos in total 102
embryos, achieved 81.37% and 84.84%,
82.05%, 76.66%, respectively. The differences
between group I and group II are not
statistically significant with p > 0.05, but
the difference between group I compared
to III and II compared with III group is
statistically significant with p < 0.05.
3. Some factors affecting the viability
of the embryo and blastocyst forming.
* The patient's age affects ability to survive
and forming blastocyst:
Of the 102 patients, we divided into 3
groups. Group I consists of 33 patients
under the age of 30 accounted for 32.35%,
group II included 39 patients aged 30 - 35
accounted for 38.24% and group III included


JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015

30 patients aged over 35 accounted for
29.41%. After 24-hour biopsy embryo survival
rate, continues to grow each group I, II and

III was 93.93%, 92.31%, 86.66%, and by
day 5 blastocyst formation rate is 84.84%,
82.05%, 76.66%. The differences between
group I and group II are not statistically
significant with p > 0.05, but the difference
between groups I and III, and II compared
to III is statistically significant with p < 0.05.
* The infertility duration affects ability
to survive and blastocyst forming:
Of the 102 embryos studied, 58 patients
having 1 - 5 years of infertility, 29 infertile
patients from 5 - 10 years, and 15 patients
above 10 years of infertility. The embryo
survival rate, continues to develop is
91.13, 93.10, 86.66 and blastocyst rate
respectively is 84.48, 82.76, 66.66. The
differences between group I and group II
are not statistically significant with p > 0.05,
but the difference between groups I and II
compared to III is statistically significant
with p < 0.05.

* Protocol of using hormone affect
embryo viability and forming blastocyst:
Of the 102 embryos studied, 53 patients
using shorter regimens, 21 patients using
long protocol and 28 patients using
antagonist protocol. The embryo survival
rate, continues to develop general is 92.45,
95.23, 85.71 and blastocyst rate is 84.90,

80.95, 75.00, respectively. The differences
between group I and group II are not
statistically significant with p > 0.05, but the
difference between groups I, II compared
to III is statistically significant with p < 0.05.
* The method of fertilization (IVF or
ICSI) affect the viability of the embryo and
blastocyst creation:
Of the 102 embryos studied, 26 patients
applied IVF, 76 patients ICSI. The embryo
survival rate, continues to develop is
88.46, 92.11 and blastocyst rate is 80.76,
81.57, respectively. The difference between
the groups is not statistically significant.
DISCUSSION

* The patient's FSH on 2 cycles affect
ability to survive and creating blastocyst:

1. Morphology before and after embryo
biopsy.

Of the 102 embryos studied, 23 patients
with hormone FSH day 2 of the menstrual
cycle < 5 IU/L, 72 patients FSH from
5 - 10 IU/L, and 7 patients with FSH over
10 IU/L. In the embryo survival rate,
continues to develop is 91.30, 91.67,
85.71 and blastocyst forming rate 84.53,
83.33, 71.42, respectively. The differences

between group I and group II are not
statistically significant with p > 0.05, but the
difference between groups I, II compared
to III is statistically significant with p < 0.05.

Embryo biopsy at day 3: In the study,
before and after embryo biopsy were
divided into 4 levels based on the
morphological structure of the evaluation
criteria of Salumets A (2001) [10]. Although
with all the comments, but measurable
number of embryonic cells and the structure
of living cells, especially those observed
cytoplasmic fragments, we can evaluate
the embryos before and after biopsy. So
far, embryo quality assessment is mainly
based on embryo morphology including cell
31


JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015

number, the ratio of cytoplasmic fragments
and uniformity between blastomeres.
Numerous studies worldwide have shown
that good quality embryos, the embryo is
uneven, with little cytoplasm fragments
was little or no morphological changes
after biopsy, the embryos have cytoplasm
fragments as much higher as decreases

the survival rate after biopsy [8]. The
research results showed that the embryo
grade 4 after biopsy is 100% intact, level
3 after biopsy is only 72.2% intact. This
result is consistent with Herhberger PE et
al research (2012), the rate of embryo
development does not increase when the
rate of cytoplasmic fragments over 20% [8].
2. Some relevant factors affecting the
ability to survive and create blastocyst.
The authors suggest that the more higher
maternal age is the lower success rate of
IVF, although on embryos of grade III and
IV but studies on the ability to continue
developing embryo, the formed blastocyst
after biopsy they found differences between
groups is still statistically significant.
For the duration of infertility, embryonic
development continues or not is largely
dependent on the quality of the early
embryo. Morphology of embryos after
biopsy does not depend on the origin of
embryos from how many year infertility
mothers but depends on the quality of the
embryos before biopsy, however in this
study the infertility duration also interfere
with the ability to survive and create
blastocyst, this study is consistent with
studies of Munne. S [9]. FSH basically not
affected to form embryos after biopsy, the

32

results are consistent with studies of
Munne et al compared the relationship
between embryo morphology and FSH
concentrations from which to assess the
relationship between FSH levels with basic
morphological changes during embryonic
development after biopsy. But for women
over the age of 35 with high FSH levels,
although embryo morphology less dependent
on FSH concentrations, but increased
concentrations of FSH associated with
reduced ovarian reserve capacity and
consistent with low rate of pregnancy in
this patients group [2]. Results between
the dosing regimens showed after embryo
biopsy and further develop and generate
blastocyst are not dependent on which is
derived from patients taking the whatever
regimen, however, for patients aged high,
prolonged duration of infertility also affects
the ability to create blastocyst, consistent
with the results of the study of Munne et al
[9].
Walker. MC, Murphy KE, Pan S (2004)
studied comparing methods of in vitro
fertilized normally (IVF) and methods
cytoplasmic sperm injection into oocytes
(ICSI) that morphology of two embryos in

two groups are the same. However, the rate
of chromosomal abnormalities significantly
higher in the patient group using method
cytoplasmic sperm injection into oocytes,
because these patients are often carry
chromosomal disorders, low sperm, weak
and deformed. But when the embryo has
reached the standard of III and IV does
not affect embryo morphology during
development after biopsy [6].


JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015

CONCLUSION
By studying 102 biopsied embryos for
PGD, we draw two conclusions:
- Laser biopsy does not alter embryo
morphology, survival and forming blastocyst.
+ After a 24-hour biopsy survival rate
of embryos, further development of the
3 groups are 93 embryos in total 102
embryos, reached 91.17%, and by day
5 blastocyst formation are 83 embryos,
reached 81.37%.
+ Cytoplasm fragment ratio, ZP thickness,
embryo diameter between groups not
change significantly.
- Several factors affect the survival and
the forming blastocyst after biopsy.

+ Maternal age, duration of infertility,
FSH levels higher are resulting decrease
of embryo survival rate.
+ Drug regimen does not influence much
embryo survival rate.
+ The method of fertilization IVF or ICSI
does not affect the survival rate and
continues to form blastocyst after biopsy.
REFERENCES
1. Trương Đình Kiệt, Hồ Mạnh Tường.
Thiết lập quy trình kỹ thuật trong chẩn đoán di
truyền tiền làm tổ (PGD) các phôi thụ tinh
trong ống nghiệm. Báo cáo đề tài cấp thành
phố 4/2011. 2011.
2. Nguyễn Thị Ngọc Phượng. Nội tiết sinh
sản. Nhà xuất bản Y học. 2013.

3. Nguyễn Viết Tiến. Dịch tễ học vô sinh
và các phương pháp điều trị. Nhà xuất bản
Y học. 2012.
4. Alfarawati. S, Fragouli E, Colls. P,
Stevens. J, Gutierrez. C, Schoolcraft. The
relationship between blastocyst morphology,
chromosomal abnormality, and embryo gender.
Fertility and Sterility. 2011, 95, pp.520-524.
5. Alikani M, Cohen J et al. Human embryo
fragmentation in vitro and its implications for
pregnancy and implantation. Fertil Steril.
1999, 71, pp.836-842.
6. Ederisinghe. WR, Murch. AR, Yovich.

JL. Cytogenetic analysis of human oocytes
and embryos in an in vitro fertilization program.
Human Reproduction. 1992, 7, pp. 230-236.
7. Ebner T, Moser M, Sommergruber M et al.
Selection based on morphological assessment of
oocytes and embryos at different stages of
preimplantation development. Hum Reprod
Update. 2003, 9, pp.251-262.
8. Hershberger PE, Gallo AM, Kavanaugh
K. The decision-making process of genetically
at-risk couples considering preimplantation
genetic diagnosis: Initial findings from a
grounded theory study. Soc Sci Med. 2012,
May, 74 (10), pp.1536-1543.
9. Munne. S, Chen S, Colls P, Garrisi J,
Zheng X, Cekleniak N. Maternal age, morphology,
development and chromosome abnormalities in
over 6000 cleavage-stage embryos. Reproductive
Biomedicine Online, 2007, 14, pp.628-634.
10. Salumets A, Hydén-Granskog C,
Suikkari AM, Tiitinen A. The predictive value
of pronuclear morphology of zygotes in the
assessment of human embryo quality. Hum
Reprod. 2001, Oct, 16 (10), pp.2177-2181.

33