Does Blastocyst Morphology Affect Live Birth Rate After Transfer of Single Euploid Embryo?

The aim of this study was to investigate whether the morphologic parameters of blastocyst inuence the live birth rate (LBR) of euploid embryos transferred in subsequent single frozen-thawed embryo transfer (FET) cycles? Methods Women who received rst preimplantation genetic testing for aneuploidy (PGT-A) and following underwent frozen-thawed single euploid blastocyst transfer cycles from June 2017 to May 2020 were divided into three age groups (< 30, 30–34 and ≥ 35 years). The primary outcome measure was LBR. Outcomes were compared between different blastocyst quality, inner cell mass (ICM) grade, trophectoderm (TE) grade and day of TE biopsy within the same age group.


Conclusion
Blastocyst quality and trophectoderm grading is a useful predictor of LBR in single frozen-thawed euploid embryo transfer cycles among women < 30 years old. However, these differences were not found in women older than 30 years.
Background Page 3/17 The development of assisted reproductive technology (ART) has greatly helped many infertile couples realize their wishes to be parents in the past few decades, while embryos suffer developmental arrest and early spontaneous abortion are still di cult problems in the treatment of infertility. In addition, with the increase of work pressure and the acceleration of life rhythm, more females choose to have children late.
Nevertheless, they will face a lower conception chance and higher miscarriage rates with age gradually increase [1] . This condition is ascribed to the rapid increase of aneuploidy rates in older women [2] .
Furthermore, it has been demonstrated that the high incidence of embryo aneuploidy is one of the important causes of in vitro fertilization embryo transfer (IVF-ET) failure [3,4] , which manifested as about 50% of embryos occurring errors during gametogenesis and early mitotic divisions throughout their preimplantation development. It is clear that the number of people who received ART treatment are rapidly aging simultaneously.
To our knowledge, conventional morphologic assessment has been a widely used method to select the embryos with the best development potential. However, it cannot accurately evaluate ploidy status, because more than half of embryos with high morphological scores were eventually screened as aneuploidy [5] . The great signi cance of ART is to achieve a healthy live birth and as the advances of molecular biology and its relative techniques, the third-generation IVF technology has been introduced.
Since then, the bene ts of PGT-A is to overcome this high aneuploidy rate through transfer of euploid embryos, which increases the likelihood of improving implantation potential and decreasing miscarriage rate from IVF therapy [6] . The current technology now used for PGT-A is based on next generation sequencing (NGS), which is done using polymerase chain reaction (PCR) ampli cation and then the results of sequencing are distinguished from normal and abnormal amounts of DNA [7] .Today PGT-A is mainly applied to the following speci c population, such as advanced maternal age (AMA), repeated implantation failure (RIF), recurrent spontaneous abortion (RSA) and severe teratozoospermia [8] .
Several studies have focused on the relationship between embryo quality and pregnancy outcomes, which did not exclude the in uence of embryo aneuploidy. Considering that the increased risk of aneuploidy is the main resistance for decline in embryo development potential, it would be reasonable to assume that as long as single euploid embryo is transferred, the higher occurrence of implantation and live birth are attained irrespective of embryo quality. However, in a retrospective cohort study by Minasi et. al. [5] , the embryos with good quality have a statistically signi cantly higher implantation rate than those with poor quality. Similarly, a recently study we published suggested that blastocyst morphologic grading was associated with implantation rate for euploid embryo transfers after adjustment for potential confounders among people under 35 years old [9] . In contrast, Anderson et al. concluded that good quality and poor quality euploid blastocysts have similar pregnancy outcomes [10] . Thus, the impact of euploid embryos quality on clinical outcomes has not been determined and are still needs further exploration. Therefore, on the basic of our previous research, the current study was conducted to identify whether the morphologic parameters of blastocysts allow for further optimization of live birth rates in transfers of known euploid embryos in single FET cycles once embryos were implanted successfully, which is helpful for doctors to select embryos to transfer and provide consultation for patients who received PGT-A for pregnancy in clinical practice.

Study design and population
This retrospective cohort study included women who undergone rst autologous PGT-A and then followed by frozen-thawed single euploid embryo transfer cycles at the Reproductive Medical Center of the Third A liated Hospital of Zhengzhou University between June 2017 and May 2020.We excluded donation cycles, women with uterine malformation and frozen oocyte cycles. This study was performed in accordance with the Code of Ethics in the Declaration of Helsinki and was approved by the Ethics Review Committee of our hospital (protocol number 2021-WZ-010).

Ovarian stimulation protocol
Each female patient usually underwent a GnRH antagonist ovarian stimulation protocol. The Gonadotropin (Gonal-F, Merck Serono, Switzerland) started injection from the second or third day of the menstrual cycle, dosage (150-300IU) was adjusted based on patient's age, basal antral follicle count (AFC), body mass index (BMI), basal follicle stimulating hormone (FSH)and ovarian reserve. The response to stimulation was assessed by performing transvaginal ultrasounds and measuring serum estradiol levels. GnRH antagonist (Cetrotide, Merck Serono, Switzerland) 0.25mg was usually injected for pituitary suppression when follicle diameter is 12 ~ 14 mm or Gonadotropin has been used for 5 ~ 6 days. GnRH agonist 0.2 mg (Dophereline, Ipsen Pharma Biotech, France) was used to trigger the nal oocyte maturation. Untrasound guided oocyte retrieval was performed 33-36 hours after the trigger.

Laboratory protocols
Blastocyst evaluation was performed prior to embryo biopsy. Blastocysts were graded according to the Gardner and Schoolcraft grading system, and the score was dependent on blastocyst expansion, ICM development and trophectoderm TE appearance [11] . The degree of expansion included the following six grades:(1) a nonexpanded embryo with the blastocele lling <50%; (2) the blastocele lling >50% of the embryo; (3) a full blastocyst with a blastocoele lling the embryo; (4) an expanded blastocyst with a blastocoele volume larger than that of the full blastocyst, with a thinning zona; (5) a hatching blastocyst with the TE starting to herniate through the zona; (6) a hatched blastocyst, with the blastocyst completely escaping from the zona. The ICM was graded as follows: (A) tightly packed, with many cells; (B)loosely gathered, with several cells; (C) very few cells. The three TE grades were: (A) many cells forming a cohesive epithelium, (B) few cells establishing a loose epithelium and (C) very few large cells. In our center, for blastocysts with an expansion score ≥4, the development of the ICM and TE was then evaluated and the ICM grade should at least B, because the ICM grade C isn't really used often. The quality of the blastocyst was grouped into three categories based on ICM and TE scoring: good quality: AA, AB and BA; average quality: BB; and poor quality: AC and BC. Embryo grading was performed by the same team of four highly trained embryologists and each with ve years of experience, which minimized the difference in human judgment. Then the embryos were biopsied on day 5 or day 6 when the expansion degree of blastocyst cavity is ³ 4. The zona pellucida was perforated by use of a Saturn laser system (Research Instruments, Singapore) to opening of 6-9 mm, and a biopsy pipette was used to aspirate 3-5 herniated TE cells. Then the washed TE cells were placed in 0.2mL PCR tubes containing 5μL phosphate-buffered saline solution (PBS). All selected embryos were screened for 24 chromosome aneuploidy with NGS, as described in Zimmerman et al [12] . Finally, three different outcomes were considered after the PGT-A testing: euploid and aneuploid and mosaic. After the biopsy, the blastocyst were vitrificated using Cryotop® (Kitazato Corporation, Shizuoka, Japan) [13] . The vitri ed-warmed procedure has been described in detailed previously [14] .

Endometrial preparation
Only embryos that were screened by NGS to be euploid were transferred in FET cycles. In general, women with regular ovulatory cycles underwent natural cycles, using transvaginal ultrasonography and urine luteinizing hormone (LH) test to monitor the development of the dominant follicle and endometrial thickness from the 10th day of the menstrual cycle until ovulation. While arti cial cycles were applied for women with irregular menses who received estradiol valerate (4~8 mg daily started on the 3rd day of the menstrual cycle) for 12 days. When endometrial thickness ≥7mm, all patients will provide for conventional luteal support and continue until 7 weeks of gestation. When on the 5th day after ovulation or the 6th day of progesterone administration, single selective frozen-thawed euploid blastocyst was transferred.

Outcome measures and statistical analysis
All statistical results were calculated with SPSS 25.0 statistical software (IBM, United States). LBR after the transfer of euploid embryos are we mainly discussed measure in this study. The secondary outcome measures are pregnancy rate and early spontaneous abortion rate. The LBR was de ned as the number of live births divided by the sum of embryos transferred cycles included in the cohort. The pregnancy rate was de ned as the percentage of the intrauterine gestational sac with fetal heartbeat by all transferred embryos at 4 weeks after blastocyst transfer. The early spontaneous abortion rate was considered to be the proportion of clinical pregnancies (a fetal heartbeat was seen on scan) that did not progress in the rst-trimester spontaneous abortion.
All cycles were divided into three groups according to the women's age (<30 ,30-34 and ≥35 years). The outcomes measure, embryos data and the baseline demographic characteristics were all compared among the three age groups. Categorical variables were compared with the Pearson chi-square (X 2 ) or Fisher's exact tests. Continuous variables were tested for normality, and they were expressed as mean ± standard deviation, and parametric data were compared using the analysis of variance (ANOVA) test. In order to further explore the association between morphologic parameters and LBR in women <30 years old, multivariable logistic regression analyses were performed. The adjusted odds ratio (aOR) with 95% con dence interval (CI) were calculated and controlled for confounding factors. P<0.05 was considered to be statistically signi cant.

Results
Finally, a total of 279 single frozen-thawed euploid embryo transfer cycles met the study inclusion  The likelihood of pregnancy rate(P = 0.410), spontaneous abortion rate (P = 0.885) as well as live birth rate (P = 0.687) were not affected by women's age between different age groups, which evaluated in Fig.  1. Table 2, the primary focus of our analysis was the LBR of different morphologic parameters related to euploid blastocysts quality between all age group. In the youngest age group (< 30 years), the prevalence of live birth was 66.67% for good quality, 65.52% for average quality and 36.36% for poor quality (P = 0.013). Nevertheless, the blastocyst quality did not affect LBR in the other two age groups. In Likewise, the effect of different TE grades was also had a relationship with LBR in youngest women (< 30 years old), which ranged from 68.75-36.36% (P = 0.012). But in women aged 30 years or older, TE grade did not in uence LBR, which ranged from 77.78-47.27% in 30-34 age group (P = 0.070) and 60.00-36.00% in patients aged more than 35 years old (P = 0.389).

As shown in
The day of TE biopsy was also associated with LBR in < 30 years old group, which was shown as 65.38% for women whose embryos were biopsied on Day 5 and 39.58% for Day 6 in (P = 0.010). However, no statistically signi cant differences were seen in the impact of the LBR on the other two age groups (P > 0.05).

Discussion
This study determined the correlation between blastocyst morphology and LBR after transfer of frozenthawed single euploid embryo. We found that euploid embryos graded as good and average are powerful predictor for LBR than euploid embryos graded as poor in women aged < 30 years. In the light of this, we further indicated that the TE grade has the great in uence on LBR. However, the LBR do not affect by euploid blastocyst morphology in women aged 30 years or older.
In general, the maternal age is one of key factors determining the possibility of pregnancy outcome either in ART conception or spontaneous conception [15] . Most importantly, the reason for age-related decline in reproductive ability is contributed to the decline of ovarian reserve function and the increase of aneuploidy with advancing women's age. And from our data, we can see the aneuploidy rate are certainly highest in older women and then start to gradually decline with women's age, although the difference is not statistically signi cant. Hence, it seems logical that we speculated the effect of maternal age on pregnancy outcome is eliminated after PGT-A. It is in line with our expectation, the pregnancy rate, spontaneous abortion rate and live birth rate were comparable between three age groups. While a literature reported that in women ≤ 35 years the chance of conception increased higher than those older 35 years old after transfer of euploid embryos [3] . In addition, we previously pointed out that if multiple embryos are euploid, morphology should be the main criterion used to select an embryo for transfer in younger women [9] . In this condition, we assume that whether blastocyst grading can foretell LBR in the frozen-thawed embryo transfer of single euploid embryo once they succeed in implantation. Given that we divided all patients into three age group to investigated whether the euploid blastocysts morphologic parameters predict the LBR in the same age groups.
To our surprise, we found that the effect of morphological parameters of euploid blastocysts is not same in different age group. There is reasonable to believe that best quality embryos have highest implantation potential and further development competence. Researches have been studied that traditional morphologic assessment has been still a guiding principle for embryo selection even among the euploid blastocysts [16,17] . Irian et al. [18] con rmed that good quality euploid embryos were associated with a higher implantation rate and LBR than poor quality euploid embryos. In their another study, they also concluded that better morphologic scores embryos yield a higher ongoing pregnancy rate compared with lower morphologic grading euploid blastocysts [19] . Consistent with their results, we also reported that the euploid embryos with higher morphologic scores had a statistically signi cantly LBR than those transfer lower morphologic scores, especially for women who younger than 30 years old after adjusting for day of TE biopsy and other possible related in uencing factor. However, there was no signi cant difference in the LBR of euploid blastocyst regardless of their morphology quality in older women. The reason for this increased prevalence of LBR may be that high quality euploid embryos of young patients have good development potential and thus result in a favorable outcome. It is suggested that morphological assessment of blastocyst may still a valuable reference when selecting embryos for transfer in the patients under 30 years who underwent PGT-A cycles. In a recent retrospectively analysis, the authors also concluded that LBR is not affected by embryo quality once PGT-A has been performed, the mean female age was 38.6±5.2 years [20] , emphasizing that the poor quality euploid embryos can also develop well. So, poor quality embryos should not be ignored, which can reduce the transfer cycles and economic burden of patients. Perhaps morphological grading alone may not be reliable due to the difference in sample size between the three age groups, we might combine time-lapse microscopy, metabonomics and protein pro les to comprehensively evaluate the quality of embryos and screen out the embryos with the most developmental potential, and thus promote successful IVF treatment outcomes.
The traditional blastocyst grading system including three morphologic parameters: the degree of blastocoel expansion, the consistency of ICM and TE. Until now, there are con icting data regarding which parameter are the most indicator to predict the outcome of blastocyst transfer. Some researchers have reported that ICM morphology can statistically signi cantly predict LBR [21,22] , because ICM is differentiated into fetal, so ICM grade should theoretically be the most important morphologic feature in uencing transfer outcomes. While recent publications in human have shown that TE quality should be corrected with viability [23,24] . This may due to TE become into the placenta, and healthy trophectoderm is required to have the capacity to invade the endometrium to initiate the complex process of implantation and to maintain normal pregnancy progress. We also found that in the younger population, embryos with TE grade B are associated with a higher LBR compared with embryos with lower morphology grading. At the same time, some researchers noted that the degree of blastocoel expansion to be a strong predictor of successful embryo implantation. Because of the very small sample size of euploid blastocysts with blastocoel expansion grades 5 and 6, we did not investigate the impact of blastocoel expansion on LBR.
It should be noted that these studies did not con rm that the blastocysts being transferred were euploid.
In contrast, Capalbo et al. [25] determined that none of the morphologic parameters provides additional valuable information for PGT-A cycles to select the best developmental embryos for transfer. Conform to our main results, the ICM and TE grade were not correlated with the LBR in women more than 30 years.
This may be due to we only included small sample size, therefore this conclusion may not represent the general population.
In addition, there is also an ongoing debate on the live birth rate of the timing of the blastulation of blastocyst. In a retrospective cohort study they reported that live birth rates were signi cantly higher with Day 6 compared with Day 5 blastocysts, regardless of embryo quality [26] . Irani et al. previously demonstrated that Day 5 blastocysts yielded a signi cantly higher LBR than Day 6 embryos of similarly graded euploid blastocysts [18] .However, our data showed that the timing of the blastulation of a euploid embryo does not in uences the LBR. The possible explanation for our results is that, morphologic grading may be a better indicator of euploid embryo development than the speed of blastulation in predicting LBR.
The strength of our research are as follows. First, all embryos and cycles were performed at a single reproductive medical center. Second, embryo scoring was conducted by the same team of four highly trained embryologists and each with ve years of experience. Third, we only transfer single euploid embryo that underwent rst autologous PGT-A treatment, this may eliminate factors which we have known can in uence our outcomes. The present study also has some limitations. First, its retrospective nature that cannot be neglected. Second, if more than one euploid embryo is available for transfer, blastocysts with good quality are usually preferred when we selected blastocysts. Thus, this may cause selection bias. Third, the number of cases in each embryo quality category were relatively small. Large prospective or sample size analysis are required to validate our current ndings in the future studies.

Conclusion
In conclusion, this study provides guidance for reproductive medical center worker that the common morphologic parameters of blastocysts assessment should be also used to help in the selection of embryos in PGT-A cycles, especially in women younger than 30 years. Furthermore, in clinical practice, we can provide consulting services for relatively older patients, if they have no good quality euploid embryos for transfer, poor quality euploid embryo are also an option, because they will produce similar LBR. All participants are exempted from informed consent to participate in this study by the Ethics Review Committee of the Third A liated Hospital of Zhengzhou University.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request

Competing interests
The authors declare that they have no competing interests Funding