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Systems Biology in Reproductive Medicine
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Good quality blastocyst from non-/monopronuclear zygote may be used for transfer during
IVF
Bao-Li Yin, Hao-Ying Hao, Ya-Nan Zhang, Duo Wei & Cui-Lian Zhang
To cite this article: Bao-Li Yin, Hao-Ying Hao, Ya-Nan Zhang, Duo Wei & Cui-Lian Zhang
(2016) Good quality blastocyst from non-/mono-pronuclear zygote may be used for
transfer during IVF, Systems Biology in Reproductive Medicine, 62:2, 139-145, DOI:
10.3109/19396368.2015.1137993
To link to this article: http://dx.doi.org/10.3109/19396368.2015.1137993
Published online: 22 Feb 2016.
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Date: 01 November 2016, At: 05:25
SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE
2016, VOL. 62, NO. 2, 139–145
http://dx.doi.org/10.3109/19396368.2015.1137993
RESEARCH ARTICLE
Good quality blastocyst from non-/mono-pronuclear zygote may be used for
transfer during IVF
Bao-Li Yin, Hao-Ying Hao, Ya-Nan Zhang, Duo Wei, and Cui-Lian Zhang
Reproductive Medicine Center, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Zhengzhou, Henan Province,
P.R. China
ABSTRACT
ARTICLE HISTORY
Although healthy infants have developed from non- and mono-pronuclear zygotes, the transfer of
embryos from non- and mono-pronuclear zygotes is not recommended because there are no
proper selection criteria. In the present study, we discuss how to select non- and mono-pronuclear
embryos with the highest developmental potential at 19–20 hours post-insemination. We found
that the percentage of blastocysts with normal chromosome constitution in non-pronuclear
zygotes was slightly higher than in mono-pronuclear zygotes. Non- and mono-pronuclear
embryos that were at the 4-cell stage on D2 and/or at the 6- to 8-cell stage on D3 had higher
incidence rates of blastocysts with normal chromosome constitutions. We also found higher
incidences of blastocysts with normal chromosome constitution on D6 than on D5. The results
suggest that if high quality non- and mono-pronuclear zygotes develop to the 4-cell stage on D2
and the 6-to 8- cell stages on D3, along with high quality D6 blastocysts, the incidence of
blastocysts with normal chromosome constitution is higher.
Received 8 July 2015
Revised 9 December 2015
Accepted 13 December 2015
Introduction
During in-vitro fertilization (IVF), the selection of embryos
with the highest implantation potential for transfer is
crucial. In the normal condition, two pronuclei and two
polar body are present at 16-18 hours (h) after
fertilization. Bi-pronuclear zygotes with good morphology
patterns are selected for further culture because these
zygotes have the highest development potential.
Meanwhile, a few zygotes present no, one, or more than
two pronuclei at 16-18h post-insemination. The non-,
mono-, tri- or poly-pronuclear zygotes are classified as
abnormal zygotes, which are discarded. However, previous
reports have shown that non-pronuclear oocytes [Burney
et al. 2008] and mono-pronuclear oocytes [Feenan and
Herbert 2006; Munne et al. 1993] can give rise to healthy
infants. Another report showed that 48.7% and 27.9% of the
embryos from mono-pronuclear oocytes inseminated by
traditional IVF or intracytoplasm sperm injection (ICSI),
respectively, were diploid and had equivalent ratios of XX
and XY chromosomes [Staessen and Van Steirteghem
1997]. Furthermore, a diploid human embryonic stem cell
line was established using mononuclear zygotes [Suss-Toby
et al. 2004], which exhibited markers of ‘stemness’ [Huan
et al. 2010]. These studies indicate that embryos defined as
KEYWORDS
Blastocysts; blastomere;
non-/mono-pronuclear
zygotes; PGS
abnormal at the pronuclear stage may have normal development potential. This is especially important for women
who do not have enough normal zygotes at the pronuclear
stage.
Noyes and colleagues [2008] analyzed the euploid
karyotypes of embryos from non- and mono-pronuclear
zygotes on day 3 post-insemination (D3) using
preimplantation genetic screening (PGS) and observed
that Y chromosomes were found in non- (17%) and
mono- (32%) pronuclear embryos, but 91% of them
were aneuploid [Noyes et al. 2008]. As a result, the transfer of abnormal pronuclear embryos in IVF is not recommended, especially when normal embryos are available.
However, non- and/or mono-pronuclear embryos
could be transferred for patients lacking bi-pronuclear
embryos, especially those with poor ovarian response.
The value of non-bi-pronuclear embryo transfer has
been discussed in a recent article by Li et al. [2015].
Until now, there are no standard criteria for selecting
embryos with the highest developmental potential in
patients without normal bi-pronuclear embryos. A few
studies have investigated the chromosomal composition
of non-bi-pronuclear embryos, but they did not discuss
how to select embryos with normal chromosomal composition. We hypothesize that non-bi-pronuclear
CONTACT Cui-Lian Zhang
[email protected]
Reproductive Medicine Center, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou
University, Zhengzhou 450003, Henan Province, P.R. China.
Color versions of one or more of the figures in the article can be found online atwww.tandfonline.com/iaan.
© 2016 Taylor & Francis
140
B.-L. YIN ET AL.
embryos with the highest development potential could be
selected by morphology and PGS. In the present study, we
investigated the chromosomal composition of blastocysts
from non-/mono-pronuclear zygotes with similar morphology to blastocysts from bi-pronuclear zygotes by
using trophectoderm biopsy and PGS.
with normal chromosomal composition was slightly
higher than that of mono-pronuclear blastocysts
(p=0.109, Figure 1C). The incidence rates of biopsied
blastocysts with Y chromosome were 56% (14/25) and
52.27% (23/44) for mono-and non-pronuclear zygotes,
respectively.
Results
The relationship between embryonic cell number
on D2 and D3, and the percentage of blastocysts
with normal chromosome constitution
Characteristics of patients
We compared the characteristics of patients between
non-, mono- and bi-pronuclear zygotes groups, and
showed that there were no significant differences
between these groups in parental BMI, parental average
age, average duration of sub-fertility, and basic folliclestimulating hormone (FSH) and luteinizing hormone (LH).
Comparison of non-/mono-pronuclear zygotes
groups to bi-pronuclear blastocysts
Of the 4,906 retrieved oocytes, 64.35% (3,157/4,906)
developed to bi-pronuclear zygotes. The incidence rates
of non- and mono-pronuclear zygotes were 7.40% (363/
4,906) and 5.61% (275/4,906), respectively (Table 1). The
incidence of blastocyst for bi-, non- and mono-pronuclear
zygotes were 45.71% (975/2,133), 30.03% (109/363), and
18.18% (50/275), respectively (Table 1). For all oocytes,
the incidence of blastocysts were 19.87% (975/4,906),
2.22% (109/4,906), and 1.02% (50/4,906), respectively,
for bi-, non- and mono-pronuclear zygotes, respectively.
The comparative genome hybridization array (aCGH)
results showed that 64.71% (44/68) and 50.0% (25/50) of
the blastocysts from non- and mono-pronuclear zygotes,
respectively, had normal chromosomal compositions
(Figure 1A, B). These rates were lower than that of
blastocysts from bi-pronuclear zygotes (69.39% (34/49)
(p = 0.59 and 0.07, non- and mono-pronuclear zygote,
respectively (IBM SPSS statistics 20, Pearson Chi-square),
Figure 1C). The percentage of non-pronuclear blastocysts
Of the embryos that reached the 4-cell stage on D2, 67.57%
(25/37) and 54.05% (20/37) of non- and mono-pronuclear
blastocysts, respectively, exhibited normal chromosomal
composition (Figure 1D, Table 2). For >4-cell embryos
on D2, the percentage of blastocysts with normal chromosomal composition was similar to those with 4-cell
embryos for non- and mono-pronuclear zygotes
(Figure 1D).
The percentage of blastocysts with normal chromosomal composition was slightly higher in embryos at the 6to 8-cell stage (76.19%, 16/21) compared to embryos at
>8-cell stage (59.57%, 28/47, p=0.185, Figure 2A,
Table 2) for non-pronuclear zygotes at D3. There was
no association between blastomere number on D3 and
the percentage of blastocysts with normal chromosomes,
for mono-pronuclear zygotes (Figure 2A).
The percentage of blastocysts with normal chromosomes were 78.95% (15/19) and 55.56% (15/27) for
embryos that developed to the 4-cell stage on, respectively
(Figure 2B, Table 2).
The incidence of blastocysts with normal
chromosome constitution on D5 and D6
We also found that the percentages of D6 blastocysts
with a normal chromosome constitution (81.82%, 18/
22, 65.22%, 15/23, Table 2) were higher than that of D5
blastocysts (56.52%, 26/46, 37.04%, 10/27; Table 2) for
non- (p=0.06) and mono-pronuclear (p=0.06) zygotes,
Table 1. Demographic characteristics of IVF-preimplantation genetic screening (PGS) patients.
Maternal average age (year)
Maternal BMI
Paternal average age (year)
Paternal BMI
Sub-fertility type
Average duration of sub-fertility (year)
Basic LH
Basic FSH
Total zygotes
Total blastocysts
Biopsied blastocysts
Non-pronuclear
28.69 ± 3.59
22.23 ± 2.77
32.54 ± 6.93
23.56 ± 3.08
Secondary infertility
3.7
5.17
6.40
363
109
68
Data presented as mean±SD besides otherwise stated.
± 3.10
± 1.41
(7.40%)
(30.03%)
(62.39%)
Mono-pronuclear
29.04 ± 4.07
22.04 ± 3.11
33.09 ± 7.30
22.66 ± 2.91
Secondary infertility
2.9
6.61
6.40
275
50
50
± 2.15
± 2.20
(5.61%)
(18.18%)
(100%)
Bi-pronuclear
31.96 ± 5.96
22.90 ± 3.17
31.00 ± 4.87
23.10 ± 2.87
Secondary infertility
3.1
5.92
8.00
3157
975
49
± 2.86
± 1.94
(64.35%)
(45.71%)
(5.03%)
P value
0.956
0.899
0.796
0.943
0.658
0.877
0.54
SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE
141
Figure 1. Chromosomal composition of abnormal zygotes. (A and B) Normal and abnormal chromosomal composition of blastocysts
from abnormal zygotes were obtained by preimplantation genetic screening (PGS). Circle: chromosome lost. (C and D) The
percentages of blastocysts with normal chromosomal composition. NP: non-pronuclear; MP: mono-pronuclear; BP: bi-pronuclear;
D2: embryo on 2 day after insemination; 4C: 4-cell stage embryo; *p < 0.05.
Table 2. Blastocyst numbers biopsied.
Non-pronuclear zygote
Day Stage
D2
4-cell
>4-cell
D3 6-8-cell
>8-cell
D5
D6
No. of Total
Blastocyst
37
31
21
47
46
22
No. of Normal
Blastocyst
25
19
16
28
26
18
Mono-pronuclear zygote
No. of Total
Blastocyst
37
13
33
17
27
23
No. of Normal
Blastocyst
20
5
17
8
10
15
Bi-pronuclear zygote
No. of Total
Blastocyst
No. of Normal
Blastocyst
28
21
22
12
D2/D3/D5/D6: embryo on 2/3/5/6 day after insemination; 4-cell/6-8-cell: embryo with 4/6-8 blastomeres.
but the differences were not significant. The percentage
of normal blastocysts on D5 (78.57%, 22/28) was
slightly higher than on D6 (57.14%, 12/21, p=0.11,
Figure 2C, Table 2) in blastocysts from bi-pronuclear
zygotes.
Discussion
In the clinical IVF setting, the transferred embryo is
selected by evaluating morphological parameters including
pronuclear, polar body, blastomere, fragmentation rate,
and cytoplasm [Scott et al. 2007]. High quality ≥4-cell
embryos on D2 and ≥7-cell embryos on D3 with early 2cell cleavage were more likely to develop into normal
blastocysts [Neuber et al. 2003]. In the present study, the
pronuclei of the embryos were checked at 19–20 h postinsemination. Embryos that developed to ≥4-cell stage on
D2, and ≥6-cell stage on D3 were subsequently cultured to
become blastocysts.
The incidence rate of diploid embryos among monopronuclear embryos has been reported as 48.7%
after IVF [Staessen and Van Steirteghem 1997]. In the
present study, our results showed that 64.71% of blastocysts from non-pronuclear zygotes with two polar
bodies, 50.94% of blastocysts from mono-pronuclear
zygotes, and 69.39% of blastocysts from bi-pronuclear
zygotes had normal numbers of chromosomes.
However, another study showed that 18% of biopsied
embryos had normal numbers of chromosomes, including 3% (1/30) of embryos from non-pronuclear zygotes,
5% (3/57) of embryos from mono-pronuclear zygotes,
and 19% (218/1,155) of embryos from bi-pronuclear
zygotes [Noyes et al. 2008]. Barbash–Hazan et al.
[2009] showed that a portion of embryos diagnosed as
aneuploidy on D3 had self-corrected on D5, and
another study observed similar results [Munne et al.
2005]. This may be the main reason that caused the
difference of incidence rates of euploid embryos from
non- and mono-pronuclear zygotes on D3 and on D5.
142
B.-L. YIN ET AL.
Figure 2. Percentages of blastocysts with normal chromosomal
composition. (A and B) The relationship between the percentage of blastocysts with normal chromosomal composition and
embryonic cell number on D3, D2, and D3; (C) the incidence
rates of D5 and D6 blastocysts with normal chromosomal
composition. NP: non-pronuclear; MP: mono-pronuclear; BP:
bi-pronuclear; D3: embryo on 3 days after insemination; D5/
D6: embryo on 5/6 days after insemination; 4C/6C/8C: 4-cell/6cell/8-cell stage embryo.
We investigated the relationship between the cell
number of embryo blastomere on D3, D2 and D3 from
non- and mono-pronuclear embryos and the incidence
of blastocysts with normal chromosome constitution.
The incidence of blastocysts with normal chromosomal
composition was slightly higher at the 4-cell stage on D2
and/or at 6-8-cell stage on D3 than that at >4-cell stage
on D2 and/or at >8-cell stage on D3. Although the
difference was not significant, the increased trend was
clear. The implantation rate of embryos transferred at
the 4-cell stage was higher than embryos that were not at
their 4-cell stage on D2 (42–44 h post-insemination)
[Scott et al. 2007]. On D3 (64–65 h), embryos transferred at the 6 to 8-cell stage resulted in better pregnancy
events [Scott et al. 2007]. Another study indicated that
there was a significant positive relationship between the
cell numbers of embryos on D2 and D3 and blastocyst
development [Neuber et al. 2003]. Therefore, we believe
that the incidence of blastocysts with normal chromosomal composition is higher if the embryos are at 4-cell
stage on D2 and/or at 6-8-cell stage on D3 for non-/
mono-pronuclear zygotes.
In the present study, trophectoderm biopsies were
performed on D5 or D6. The results showed that the
percentage of normal blastocysts was higher on D6 than
on D5, especially for mono-pronuclear zygotes, although
the results did not reach significance. We found that the
incidence of D5 blastocysts with normal chromosomal
constitution was higher than D6 blastocysts for bi-pronuclear zygotes. Previous studies indicated that the
majority of normally fertilized zygotes were bi-pronuclear
at 16–18 h post-insemination [Nagy et al. 1998; Rienzi
et al. 2005], and embryos developed to the hatching
blastocysts stage on D5 [Kirkegaard et al. 2013]. When
the pronuclear zygotes were reanalyzed 4–6 h later, however, 25% of the mono-pronuclear zygotes at 16–18 h
post-insemination were found to have developed a second
pronucleus [Staessen and Van Steirteghem 1997]. These
findings indicate that embryonic development may be
delayed for non- and mono-pronuclear zygotes compared
with di-pronuclear zygotes.
Another possible explanation for the formation of
non-pronuclear zygotes includes the accelerated breakdown of pronuclear membranes and a lack of visualization due to granularity [Manor et al. 1996; Munne and
Cohen 1998]. Generally, pronuclei are checked at 16–18
h post-insemination. Previous studies indicated that the
breakdown of pronuclear membranes occurred at
21–24 h post-insemination [Chamayou et al. 2013;
Kirkegaard, et al. 2013]. In the present study, the
pronuclei were checked at 19–20 h post-insemination.
Thus, the normally fertilized oocytes may be disregarded in the present study. This may be a reason for
the higher incidence of normal blastocysts for nonpronuclear zygotes compared with mono-pronuclear
zygotes.
Although healthy children from non- and mono-pronuclear zygotes have been born, the transfer of an abnormal embryo with good morphology is still not
recommended. We found that D6 blastocysts of nonand mono-pronuclear zygotes had higher incidence rates
of normal chromosomal composition, although the difference is not significant for mono-pronuclear zygotes,
which may be caused by the limited number of biopsied
samples. There is still a debate about the use of PGS for
the detection of chromosome aneuploidy in embryos
due to the common mosaicism in trophectoderm cells.
SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE
Furthermore, there is limited research on the morphology of healthy embryos [Palini et al. 2015]. We did not
investigate the implantation rate and clinical parameter
for these blastocysts. Thus, further studies are needed
before ‘abnormal embryos’ can be used for transfer in
the clinic.
In this study, we used array CGH to screen whole
chromosomes. Although CGH has many advantages
compared to fluorescent in situ hybridization (FISH)
and is a gold standard [Handyside 2013] to identify
aneuploid embryos in the clinic, it cannot detect whole
ploidy errors for example distinguishing haploid,
triploid, and tetraploid cells from diploid ones [Wilton
2005]. Therefore, we removed the zona pellucida before
biopsy and washed three times using medium. We
further analyzed the rate of Y chromosome blastocysts
in each group. The result indicated that our samples
were not contaminated by cumulus cells.
Since cases of normal infants born from mono- and
non-pronuclear zygotes [Burney et al. 2008; Feenan and
Herbert 2006; Munne et al. 1993] are reported and diploid
human embryonic stem cells are established using monopronuclear zygotes [Huan et al. 2010; Suss-Toby et al.
2004], a few other studies have confirmed that a part of
non-bi-pronuclear zygotes have normal chromosome
constitution and can develop to blastocyst [Noyes et al.
2008; Staessen and Van Steirteghem 1997]. This means
that a few women without available bi-pronuclear
embryos during an IVF cycle can obtain a baby using
mono- or non-pronuclear embryos which is especially
interesting for poor ovarian response women. However,
there is no study giving an appropriate way to select the
available mono- and non-pronuclear zygotes in clinic. In
the present study, we found that the incidence rates of
blastocysts with normal chromosomal composition were
higher for non- and mono-pronuclear embryos at the 4cell stage on D2 and/or at the 6- to 8-cell stage on D3, and
on D6 than on D5. However, the differences were not of
high significance, and more studies are needed to determine the possible use of abnormal zygotes in clinics.
Although our findings still cannot be used as an appropriate way to select available embryos from mono- and
non-pronuclear zygotes in clinic, they may be helpful to
establish a better method in the future.
Materials and methods
Patients
This prospective study was supported by the Ethics
Committee of Zhengzhou University. A total of 788 IVF
fresh cycles in the Reproductive Medicine Center of
People’s Hospital of Zhengzhou University from July,
143
2014 to October, 2014 were enrolled in this study.
Written informed consents were obtained from all subjects. All patients had normal FSH level during early
follicular phase, fewer than three previous treatment
cycles, and a negative test result for hepatitis and HIV.
We excluded patients with polycystic ovaries, obesity, and
other diseases that may affect oocyte quality. Women of
various ages (Table 1), and those receiving different ovarian stimulation protocols were included.
Embryos
Ovulation was induced by administering 10,000 UI
of human chorionic gonadotrophin (hCG, Pregnyl
from Organon, Holland, or Profasi from Serono,
Switzerland). Oocytes were retrieved by an ultrasoundguided method at 36–37 h after hCG administration.
The cumulus-oocyte complexes (COC) were washed
with G-MOPS (Vitrolife, Sweden) and incubated overnight at 37°C. Then COCs were incubated in G-IVF
(Vitrolife) with humidified tri-gas (5% CO2, 5% O2,
and 90% N2) for 2 h at 37°C covered with oil.
Traditional IVF was carried out in G-IVF medium for
4 h. If the second polar body was observed under microscope, we defined the oocyte as fertilized. At 19–20 h
post-insemination, pronuclei were checked. Pronuclei
and embryonic morphology were assessed according to
previous studies [Scott and Smith 1998; Steer et al. 1992;
Tesarik and Greco 1999]. Only one or none of the pronuclear zygotes with two polar bodies were selected and
classified as abnormal zygotes. In the present study,
embryos were fertilized using IVF (not ICSI), and the
abnormal zygotes were incubated in humidified tri-gas
in G1 (Vitrolife) medium droplets covered with oil at
37°C. If embryos developed to ≥4 cells stage on D2, ≥6
cells stage on D3, and fragmentation rate ≤20%, the
embryos were incubated in G2 (Vitrolife) droplets
covered with oil until day D5 or D6 post-insemination. The blastocysts were assessed according to the
description of Gardner [Balaban 2011; Gardner and
Schoolcraft 1999]. Briefly, the blastocyst was divided
into six grades (1 to 6) according to stage of development: 1, early; 2, blastocyst; 3, blastocyst with partly
expanded blastocoel; 4, blastocyst with completely
expanded blastocoel; 5, partly hatched/hatching; 6
completely hatched. According to inner cell mass
(ICM), blastocysts were divided into three grades: A,
tightly packed ICM with many cells; B, a loosely
grouped ICM with many cells, and C, ICM with very
few cells. For trophectoderm (TE), blastocysts were
divided into A, B, and C: A, many cells forming a
cohesive epithelium; B, few cells forming a loose
epithelium; C, very few cells. The blastocysts which
144
B.-L. YIN ET AL.
developed to the 4-6 stage, ICM scored ≥B grade, and
TE scored ≥B grade were defined as good blastocysts.
The high quality D5 or D6 blastocysts were then
biopsied. Before biopsy, the zona pellucida was
removed and washed three time using G2 medium.
The samples were pooled together and stored at -80°C
until used. Amplified genome DNA samples were
randomly selected. All mono-pronuclear blastocysts
were biopsied because only 50 blastocysts met quality
criteria. The biopsied TE cells were washed using
1×PBS and put in PCR tubes with 2.5 μl PBS.
Samples were stored at -20°C until used.
PGS procedure
Whole genomic amplification and aCGH were performed
as previously described [Liu et al. 2012; Yang et al. 2012].
Briefly, whole genomic amplification was carried out by
using the Sure Plex kit (BlueGnome, Cambridge, UK)
according to the manufacturer’s protocol. The amplified
samples and controls (normal male and female)
were labeled with Cy3 and Cy5 fluorophores for 2–4 h at
37°C. Labeled DNA was re-suspended in dexsulphate
hybridization buffer, and hybridized with 24 sure chips
(BlueGnome) for 4–6 h. The chips were then washed
four times and dried. Dried chips were scanned using a
laser scanner (Agilent, Santa Rosa, CA, USA). Scan
data were analyzed using the BlueFuse Multi software
(BlueGnome).
Statistical analysis
Differences between groups were tested by chi-square
test. P<0.05 was considered significantly different.
Acknowledgments
We appreciate all the members in the Reproductive
Medicine Center, People’s Hospital of Zhengzhou
University for their contributions.
Declaration of interest
This study was supported by Henan Academy of medical
science and technology plan (201202002). The authors
declare no conflicts of interest.
Notes on contributors
Designed the assay and acquired data: YBL, ZCL;
Contributed to data acquisition: HHY, ZYN, WD; Analyzed
data and drafted the article: GZJ.
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