Background: This retrospective cohort study
aims to determine the relationship between morphologic grading of day 1
or 3 embryos and euploid blastocyst rate in the preimplantation genetic testing
cycle. Methods: 2001 two pronucleus (2PN) embryos were obtained from 219
patients in our in vitro fertilization center on day 1. Embryo
morphologic grading was conducted on day 1 and day 3. A blastocyst trophectoderm
biopsy was conducted on day 5 or day 6, followed by aneuploid screening using
next a generation sequencing platform. Chi-square test, Student’s
t-tests and Mann-Whitney U tests were used to compare
categorical and continuous variables between the cohorts. Logistic regression
analysis for euploidy was conducted to determine the association of embryo
morphological grading with blastocyst euploidy. Results: 811 blastocysts
selected from 2001 2PN embryos were appropriate for biopsy and DNA from all
biopsies were successfully amplified for aneuploidy screening. The day 1
pronuclear pattern showed a weak, non-statistically significant association with
euploid blastocyst (p
In past 40 years, in vitro fertilization (IVF) success rates have remarkably improved [1, 2, 3]. However, analysis of embryonic developmental potential remains a significant challenge [4, 5, 6]. Chromosomal abnormality is the most common cause for miscarriage, and selection of euploid embryos for implantation can markedly improve IVF outcomes [7, 8]. Morphologic assessment is the primary method for embryo selection, the factors used in assessment include oocyte, zygote, cleavage-stage embryo, and blastocyst. However, this approach cannot evaluate the status of the embryonic genome [9, 10, 11, 12]. Time-lapse microscopy (TLM) can monitor embryo development over a 24 hour window without removing the embryo from the incubator. Due to recently acquired knowledge of embryo development dynamics, TLM technology has the potential to become a priority method for embryo selection. However, concerns are that the needed equipment is expensive and there is potential harm from 24 hour monitoring of the embryo, thus the use of TLM remains controversial. Moreover, the cost of equipment and consumables will increase the economic burden on patients seeking IVF [13, 14, 15, 16].
Preimplantation genetic testing for aneuploidy (PGT-A) reduces the time to pregnancy and avoids the transfer of aneuploidy embryos [17, 18, 19, 20]. Early on, IVF centers used blastomere biopsy on day 3 to identify euploid embryos . Within the development of blastocysts in culture, vitrification, trophectoderm (TE) biopsy, and next generation sequencing (NGS) are wildly used for aneuploid testing [22, 23]. Although PGT-A has many advantages for patients, damage to the trophectoderm (TE) from embryo biopsy is still unclear , and some studies considered that TE plays a crucial role during embryo development [10, 25]. Several studies have demonstrated that blastocyst grading can predict euploid blastocysts, but the association is weak or moderate [26, 27]. Liu et al.  have studied the correlation between day 3 morphologic grading and pregnancy outcomes in preimplantation genetic testing (PGT) cycles that underwent blastomere biopsy and found that poor/fair-quality embryos have a substantially reduced likelihood of retaining viability after biopsy. However, studies that have explored the potential correlation between day 3 morphologic grading and euploid blastocysts which have undergone trophectoderm (TE) biopsy remains still limited.
A total of 219 women who have at least one blastocyst available for biopsy participated in this study. 811 biopsied blastocysts obtained from 2001 embryos underwent aneuploidy screening using a NGS platform, and on days 1 and 3 morphologic grading was recorded. We observed that morphologic grading on day 3, but not day 1, was a significant predictor for euploid embryo identification.
219 PGT cycles which come from 219 patients were performed between 2017 and 2019
at our IVF center included in this retrospective cohort study. Patients in PGT cycle who have obtained at least one biopsied blastocyst and agreed to participate this study were included. Patients did not obtained any biopsied blastocysts and refused to participate this study were excluded. Total 2001 two
pronucleus (2PN) embryos formed after intracytoplasmic sperm injection (ICSI) and
all the 2PN embryos were assigned for blastocyst culturing. At last, 811 biopsied
blastocysts were obtained from 219 women. All patients who participated in this
study were divided into 3 groups, PGT-A, preimplantation
genetic testing for monogenic (PGT-M) and preimplantation genetic testing for
structural rearrangements (PGT-SR), based on the results of genetic counselling.
PGT-A patients underwent at least two spontaneous miscarriages or at least once
miscarriage indicating abnormalities in chromosomal structure. The mean age of
patients assigned to PGT-A was 38 years old and had implantation failure
|54 patients||91 patients||74 patients||219 patients|
|519 2PN embryos||976 2PN embryos||506 2PN embryos||2001 2PN embryos|
|FSH ( IU/L)||5.7
|LH ( IU/L)||3.5
All were oocytes placed in Quinn’s Advantage Fertilization Medium (ART-1020,
Origio, Pasadena, CA, USA) supplemented with 5% human serum albumin (HSA)
(90165, Irvine Scientific, Santa Ana, CA, USA) under oil (10029
Ovoil, Vitrolife, Gothenburg, Sweden). Intracytoplasmic sperm
injection (ICSI) was conducted approximately 4 hours after oocyte retrieval and
after this, oocytes were returned to the incubator for culture. Embryos were
cultured up to the blastocyst stage in 6% CO
All embryo assessment followed the Istanbul consensus or Gardner’s
system for grading human blastocysts. Zygote assessment was performed at 17
Blastocyst quality was assessed prior to TE biopsy. Only blastocyst
Student’s t-tests or Mann-Whitney U tests were conducted to
assess statistically significant differences. Continuous variables are shown as
From January, 2017 to December, 2019, data from 2001 2PN embryos which originated from 219 patients, in 219 PGT cycle, were included in this study. The 219 patients and 2001 2PN embryos were divided into 3 groups based on data gathered. Specifically, 54 patients and 519 embryos were placed in the PGT-M group, 91 patients and 976 embryos were placed in the PGT-SR group, and 74 patients and 506 embryos were placed in the PGT-SR group. IVF-PGT cycle information and patient demographics are outlined in Table 1.
We assessed the effects of different morphological factors on blastocyst
euploidy using logistical regression analysis, and measured euploid blastocyst
development at day 5 or day 6. In day 3 blastocysts, blastomere number, symmetry
grading, and fragmentation grading were included in the logistic repression
analysis. The results outlined in Table 2 indicate that blastomere number had the
strongest association with blastocyst euploidy (odds ratio (OR) = 1.156, 95% CI
= 1.103–1.121, p
|Parameter||OR||95% CI||p value|
We next analyzed the association between blastomere number and euploid
blastocysts in the three PGT groups. Embryos were divided into 9 sub-groups based
on blastomere number in each group (Fig. 1A). In the PGT-M group, 9 day 3
blastomere embryos obtained the highest rate of euploidy, followed by
Univariate analysis of day 3 morphological grading factors in PGT embryos. Univariate analysis of day 3 embryo blastomere number (A), symmetry grading (B) and fragmentation grading (C) in PGT-M (red), PGT-SR (blue), PGT-A (green) and PGT total (black).
|Blastomere number||Embryo number||Euploid number||Euploid rate||Embryo number||Euploid number||Euploid rate||Embryo number||Euploid number||Euploid rate||Embryo number||Euploid number||Euploid rate|
|Symmetry||Embryo number||Euploid number||Euploid rate||Embryo number||Euploid number||Euploid rate||Embryo number||Euploid number||Euploid rate||Embryo number||Euploid number||Euploid rate|
|Fragmentation||Embryo number||Euploid number||Euploid rate||Embryo number||Euploid number||Euploid rate||Embryo number||Euploid number||Euploid rate||Embryo number||Euploid number||Euploid rate|
Embryo selection and euploid embryo transplantation are critical steps for
improving pregnancy rates following IVF. In this study, 811 biopsied blastocysts,
which were obtained from 2001 2PN embryos in 219 patients, were included in our
analysis. As outlined, patients were divided into 3 groups, PGT-M, PGT-SR and
PGT-A based on clinical indications. We found several euploid embryo predictors
within IVF preimplantation genetic testing (PGT) morphologic data. Day 5 or day 6
blastocysts with quality
Maternal age is a critical factor for blastocyst euploidy. This factor can affect the blastocyst euploid rate . Advanced age patients in IVF treatment usually obtained lower quality embryos and clinical pregnancy rate and higher miscarriage rate than the younger patients . Average percent euploid embryos increased from ~60% to ~75% between maternal ages 22 and 28, dipping to ~60% by age 35, followed by a steady decline to ~40% by age 40 until reaching ~10% by age 45 [36, 37, 38]. In our study, the maternal age distribution in these 3 groups is significantly different and results showed that PGT-A group being the oldest (Table 1). Otherwise, chromosome abnormality is also a factor can not be ignored in blastocyst euploidy. Such as Balanced translocations, Robertsonian translocations, insertions, and inversions are abnormalities that change the natural order of chromosomal segments. The carriers of above abnormalities are typically asymptomatic but more easily produce chromosomal copy number abnormal gamete. This result will make infertility problems, increase the possibility of miscarriage, fetal anomalies and affect offspring’s intelligence [39, 40, 41]. On the other hand, most of patients in PGT-M group do not have the infertility, and their blastocyst have relative high euploid rate. So, we analyzed the results in different group to avoid the impact of different group clinical indications on the final results (Table 1).
We examined the effect of blastomere number on predicting embryonic euploidy in
three groups. 9 blastomere embryos received the highest euploidy rate in the
PGT-M group, but 8 blastomere embryos received the highest euploidy rate in the
PGT-SR and PGT-A groups. It is commonly viewed that 7–9 blastomere embryos are
considered the best choice for transplantation; however, in this study embryos
Recently, a variety of technologies for euploid blastocyst screening have been developed. Preimplantation genetic testing for aneuploidy is an attractive technology that has the potential to increase IVF success rates . However, the association of TE or blastomere biopsy and impairment of embryonic development remains controversial, as well as biopsy increasing the time exposure out of incubator [18, 19]. Day 5 or day 6 blastocyst scoring has been confirmed as a feasible method for embryo selection, but blastocyst scoring cannot be applied to embryos only culture for 3 days, and approximately 40% of embryos fail to reach blastocyst stage. Alternatively, day 3 embryo scoring systems involve more variables than the blastocyst scoring system, so day 3 embryo assessment likely offers more morphologic information than either day 5 or day 6 embryos.
Time-lapse microscopy is a novel technology for optimizing embryo selection. Many studies have demonstrated that some parameters of embryo kinetics are useful for embryo selection, but a gold standard in the field is still lacking [46, 47, 48]. Potential embryo damage from exposure to camera lamplight every 5 mins remains unclear [13, 14, 15]. Recent attention has focused on developing non-invasive approaches for PGT such as analysis of cell-free DNA in blastocoelic fluid or culture medium . Although noninvasive methods decrease embryo impairment, the accuracy and specificity of these noninvasive approaches require improvement [50, 51].
There are a number of limitations in this study. Additional data is required to confirm the benefit by applying the conclusion of our study for the IVF cycle. We would further assess the impact of day 3 embryo morphology on the rates of clinical pregnancy, implantation, miscarriage, and live birth. We found as showed in Table 1 that the euploid embryo number per patient in PGT-M group is less than PGT-A group. The main reason for this finding may be the limited sample size.
In conclusion, this study confirmed that day 3 embryonic blastomere number,
symmetry, and fragmentation are statistically significant predictors of euploid
blastocysts. In contrast, the 2PN pattern in day 1 embryos was not associated
with blastocyst euploidy. 8 blastomere or blastomere number
The data that support the findings of this study are available from Guangzhou Women and Children’s Medical Center but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Guangzhou Women and Children’s Medical Center.
YL designed the research study. YL, ZO and ZC performed the research. ZO provided help and advice on data collection. YL, ZO and ZC analyzed the data. YL and ZO wrote the first draft of the paper. ZC provided critical reviews and interpretation of the results. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by Reproductive Medical Ethics Committee of Guangzhou Women and Children’s Medical Center (Approval number: 2023-152A01) and written informed consent was obtained from all subjects.
We thank all the staffs from the Center of Reproductive medical, Guangzhou Women and Children’s Medical Center for help and support. We also appreciate the patients who participated the study.
This study was funded by Guangzhou Women and Children’s Medical Center (YIP-046).
The authors declare no conflict of interest.
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