Background
In vitro fertilization-embryo transfer (IVF-ET) refers to an assisted reproductive technology in which gametes (sperms and eggs) are collected from ovaries and fertilized under in vitro conditions to form embryos, and then high-quality embryos are implanted in a uterus to develop into foetuses. To date, more than eight million babies have been born worldwide as a result of IVF-ET. Multiembryo transfer leads to a high multiple pregnancy rate up to 20% [
1]. Multiple pregnancies may increase the risk of adverse pregnancy outcomes and endanger maternal and infant health. Elective single embryo transfer (eSET) has been increasingly used worldwide as the most effective method to reduce the rate of multiple pregnancies [
2]. However, the success rate of SET has been limited mainly by the lack of scientific methods evaluating the developmental potential of embryos.
Currently, the most commonly used method for embryo selection is morphological assessment. However, nearly 50% of embryos with good morphology are aneuploidy, suggesting that morphology alone is insufficient for chromosomal assessments of embryos [
3,
4]. Embryonic aneuploidy is an important cause that decreases the pregnancy rate and increases the miscarriage rate in IVF. Studies have confirmed that aneuploidy can cause developmental arrest and implantation failure of embryos [
5]. Embryonic aneuploidies are responsible for more than 50% of abortions [
6]. Clinically, for patients with a high risk of producing aneuploidy embryos, such as women of advanced age and women who have experienced recurrent miscarriages or multiple implantation failures [
7,
8], an approach that helps avoid aneuploid embryo transfer is using preimplantation genetic testing for aneuploidy (PGT-A) to analyse the chromosome copy number before implantation. However, for patients who did not undergo PGT-A in a fresh cycle and had implantation failure or miscarriage after fresh embryo transfer, the only option is to select frozen embryos based on morphological assessment, which cannot determine the status of chromosomes. For these patients, PGT-A of frozen embryos requires a series of procedures including thawing, biopsy, and refreezing. In particular, embryo biopsy is invasive. Embryo biopsy requires special equipment and well-trained professionals and may have a negative impact on the embryo’s ability to develop and implant [
9,
10]. In addition, a long-term follow-up must be performed due to offspring safety concerns related to the biopsy cycle [
11,
12]. Therefore, if the chromosome ploidy of embryos can be detected by non-invasive chromosome screening (NICS), the embryo biopsy will be avoided after thawing, which reduces the possibility of embryo damage.
Since Stigliani et al. [
13‐
16] first discovered that embryos release cell‐free DNA (cfDNA) into culture medium during culture, noninvasive PGT-A using cfDNA has become a research hotspot in the field of assisted reproduction. In particular, studies on the culture media of frozen-thawed embryos showed that compared with fresh embryo culture medium, frozen embryo culture medium was a more suitable material for niPGT-A. In previous studies, after frozen embryos were thawed and cultured for 14–24 h, the culture medium or a mixture of blastocoel fluid and culture medium were collected and yielded a cfDNA amplification success rate of 92.3–100%. Taking the whole-embryo results as a gold standard for comparison, the results from culture medium were highly concordant and reached an accuracy of 87–100% [
17‐
20]. Kuznyetsovet al. [
17] reported that the concordance rate between noninvasive chromosomal screening results and whole-embryo results was even higher than that between trophectoderm (TE) results and whole-embryo results (96.4% versus 87.5%). However, the duration of embryo thawing and culture was relatively long (14–24 h) in the studies above compared to that in frozen-thawed cycles (generally two to three hours) [
21,
22]. In addition, considering that a test for copy number variation (CNV) requires 9 h [
23], the total in vitro culture time would be 23–33 h, which is too long for frozen blastocysts that have met the freezing requirements to be transferred within the optimal time window. Therefore, the approach applied in the studies above may reduce the embryo implantation success rate and is not suitable for application in clinical practice.
In this observational study, a clinically implementable embryo thawing and culture method for single-blastocyst transfer (SBT) was used, where frozen embryos selected according to morphological grades were thawed and cultured in 15–20 µL of culture medium for 6 h. The patients were followed up for clinical outcome evaluations. Meanwhile, the culture media of blastocysts were collected for NICS, and the relationship between NICS results and the clinical outcomes of patients was compared to explore whether NICS results can be used to effectively assess the developmental potential of frozen-thawed embryos. The present study included 212 IVF or ICSI patients who underwent frozen-thawed SBT at our centre and represents the first large-scale retrospective study analysing the relationship between NICS results and clinical outcomes in frozen-thawed SBT.
Discussion
In this study, we retrospectively analysed and compared the NICS data of 210 frozen-thawed blastocyst culture medium samples and the corresponding clinical outcomes of patients who received SBT with morphologically good-quality embryos. The results showed that the euploidy group had significantly higher clinical pregnancy, ongoing pregnancy, and live birth rates than the aneuploid group (56.2% versus 29.4%, adjusted OR 0.33, 95% CI 0.15–0.72; 47.2% versus 22.1%, adjusted OR 0.34, 95% CI 0.15–0.77; 46.1% versus 22.1%, adjusted OR 0.39, 95% CI 0.18–0.86, respectively), but this group showed nonsignificant differences in the three parameters compared with the chaotic abnormal/NA embryo group (56.2% versus 60.4%, adjusted or 0.85, 95% CI 0.39–1.85; 47.2% versus 49.1%, adjusted or 0.76, 95% CI 0.36–1.63; 46.1% versus 49.1%, adjusted or 0.78, 95% CI 0.37–1.67, respectively) (Fig.
2 and Additional file
1: Table S2), suggesting that the patients who were implanted with euploid embryos had more satisfactory clinical outcomes than those implanted with aneuploid embryos, which is consistent with findings from previous studies [
31,
32]. However, the aneuploidy group had a live birth rate of 22.1%, indicating that either the test results were false positive or the embryos had the ability to repair themselves. In addition, the chaotic abnormal/NA embryo group was not significantly different from the euploidy group in clinical pregnancy, ongoing pregnancy, and live birth rates (56.2% versus 60.4%, adjusted or 0.85, 95% CI 0.39–1.85; 47.2% versus 49.1%, adjusted or 0.76, 95% CI 0.36–1.63; 46.1% versus 49.1%, adjusted or 0.78, 95% CI 0.37–1.67, respectively), possibly because this group had a high proportion of morphologically ‘good’ embryos (good: 54.7%; fair:37.1%; poor:25.0%) (Table
4). A ‘good’ embryo has a dense cell arrangement and might release less DNA into culture medium, resulting in test failure or indeterminate results. Magli et al. [
33] also found that transferring an embryo with successful blastocoel fluid amplification led to a clinical pregnancy rate of only 37% and an ongoing pregnancy rate of 18%, while transferring an embryo with blastocoel fluid amplification failure resulted in a clinical pregnancy rate of 77% and an ongoing pregnancy rate of 70%. Therefore, in cases where euploid embryos are unavailable, a possible solution is to consider embryos with high morphological quality but test failure by sequencing if the patient provides informed consent after fully understanding the risk.
In this study, frozen embryos were thawed and cultured following a routine protocol in our centre, i.e., thawed blastocysts were cultured in microdrops (15–20 µL) for 6 h, and SCMs were collected for embryo chromosomal genetic testing. In a previous study conducted by Kuznyetsov et al. [
17], a mixture of blastocoel fluid and culture medium collected after frozen embryos were thawed and cultured in a 25 µL culture system for 24 h had an amplification success rate of 100%, and the NICS results had a concordance rate of 96.4% with whole-embryo tests. In the study of Huang et al. [
18], in which frozen embryos were thawed and cultured in a 15 µL culture system for 24 h, the SCMs had an amplification efficiency of 92.3%, and the NICS results had a concordance rate of 93.8% with whole-embryo tests. Jiao et al. [
19] reported that after frozen embryos were thawed and cultured in a 12-µL culture system for 15 h, a mixture of blastocoel fluid and culture medium had an amplification efficiency of 100%, and the NICS results had a concordance rate of 90.48% with whole-embryo tests. In a recent study by Li et al. [
20], after 41 frozen embryos classified as mosaics were thawed and cultured in a 15 µL culture system for 14–18 h, the culture medium, TE cells, and remaining whole embryos were collected for NICS and PGT-A. The results showed that 85.4% of the whole embryos were euploidy, 82.9% of which were reported to be euploidy by NICS [
20]. The studies above showed that niPGT-A has the potential for embryo chromosomal screening. However, in clinical practice, the total thawing and culture duration of frozen embryos is generally 2–3 h [
21,
34] such that an embryo that has developed into a blastocyst can be implanted within the optimal time window to improve the success rate of implantation. In the present study, we thawed and cultured frozen embryos for 6 h following a routine protocol in our centre, which can fully activate the developmental potential of frozen-thawed embryos, facilitate embryo implantation, and meet the sample size requirement for NICS.
Before the sequencing depth of the samples was confirmed, the raw reads of 53 samples at different depths were analyzed. The sequencing reads were reduced to: 200 K, 300 K, 400 K, 500 K, 600 K, 800 K, 1 M. Among the 53 samples with ≥ 10 Mb duplications or deletions, when the reads reached 400 k or more, the CNV results obtained by the analysis are consistent for the same sample. The consistency rate is 100% (53/53). Among the 29 samples with ≥ 10 Mb duplications or deletions and 30–70% mosaicism, when the reads reached 400 K or more, the CNV results obtained by the analysis are consistent for the same sample. The consistency rate is also 100% (29/29) (Additional file
2: Figure S1). These results showed that the CNV accuracy can be credible when the amount of sequencing data for each sample must reach 400 K. In addition, we also refer to the sequencing depth of the recently published articles on NICS [
19,
20,
25‐
27]. Finally, the sequencing reads in this study were confirmed to be ~ 2 M.
To further explore the factors affecting clinical outcomes, we performed stratified analyses for female age, morphological grade, and embryonic days. The results showed that the embryo ploidy rate decreased with increasing female age (45.2% in the < 35 age group and 31.0% in the ≥ 35 age group), and the clinical pregnancy, ongoing pregnancy, and live birth rates also showed similar declines (55.4% versus 21.4%, 45.8% versus 14.3%, 45.2% versus 14.3%, respectively) (Table
2), which is consistent with the results from previous studies [
31,
32]. Compared with D6 blastocysts, D5 blastocysts did not show any significant difference in the euploidy rate (40.4% versus 48.1%,
p = 0.320) but resulted in significantly increased clinical pregnancy, ongoing pregnancy, and live birth rates (57.7% versus 22.2%; 48.1% versus 14.8%; 48.1% versus 13.0%, respectively) (Table
5). Kovalevsky et al. [
35] reported that patients with D5 frozen embryo transfer had significantly higher clinical pregnancy and ongoing pregnancy rates than those implanted with D6 frozen embryos. In the present study, D5 embryos had a slightly lower euploidy rate (40.4% versus 48.1%) but a higher chaotic/NA rate than D6 embryos (30.1% versus 11.1%), possibly because in D5 embryos, good embryos were significantly higher than D6 (p < 0.001, Additional file
1: Table S9). Good embryos have dense cells and release less DNA into culture medium during thawing and warming, leading to a higher chaotic abnomal/NA rate.
Based on the NICS results, the percentage of aneuploid embryos was higher among the embryos with a worse morphology, as evidenced by the data showing that aneuploid embryos accounted for 21.5% of morphologically good-quality embryos, 34.6% of fair-quality embryos, and 46.0% of poor-quality embryos(
P = 0.013).The overall clinical pregnancy, ongoing pregnancy, and live birth rates of patients decreased with morphological quality deterioration of embryos (59.5% versus 44.4% versus 38.0%,
P = 0.038; 49.4%versus 39.5% versus 24.0%,
P = 0.016; 49.4%versus 39.5% versus 22.0%,
P = 0.008) (Table
3). Peng et al. [
36] found that the morphological development of embryos had a positive effect on the pregnancy and live birth rates but not on miscarriage rates in patients with euploid embryo transfer. In the present study, the live birth rate was highest (57.6%) in patients implanted with morphologically ‘good’ euploid embryos (Table
3). Therefore, euploid embryos with a ‘good’ morphology should be the first choice for implantation. According to the live birth rate, we recommend that the ideal embryo for transfer is ‘euploid embryo with good morphology’, followed sequentially by ‘chaotic abnormal/NA embryos with good morphology’, ‘euploid embryo with fair morphology’, and ‘chaotic abnormal/NA embryos with fair morphology’ (Table
3).
In addition, this study included IVF-fertilized embryos. Previous research has shown that the lysis conditions for WGA of biopsied cells (polar bodies, blastomeres, or TE cells) might be too mild to amplify sperm DNA, whereas the amplification of single-sperm DNA requires strong lysis conditions, and therefore, PGT might be suitable for IVF-fertilized embryos [
37]. In the present study, no significant difference in clinical outcomes was found between patients who received ICSI and IVF. De Munck et al. [
38] also reported that PGT-A did not result in any differences in the blastocyst formation rate, total number of blastocysts, and euploidy rate between embryos fertilized by ICSI and IVF. Therefore, niPGT-A may be applicable regardless of fertilization approaches such that both fertilization procedures and chromosomal screening will be noninvasive in the future.
This study also has some limitations. To avoid the impact of refreezing on the embryos, the embryos must be implanted during the current frozen-thawed cycle. In our protocol, the estimated time for thawing, testing, and implanting is at least 15 h. Therefore, our method is most applicable for early blastocysts frozen in stage 3 or 4, which allows sufficient time for testing, and then the blastocysts can be transferred directly without being frozen again. However, the method is not recommended for blastocysts frozen at stages 5–6. The long culture time might cause the embryos that have developed into blastocysts to miss the optimal time window for implantation and embryo hatching, leading to a lower success rate of implantation.
In conclusion, this study is the first large-scale retrospective clinical study to analyse the relationship between NICS results and clinical outcomes in patients implanted with single frozen-thawed blastocysts selected based on morphological quality. The results showed that in clinical practice, frozen embryos could be thawed and cultured in 15–20 µL of culture medium for 6 h, and the culture medium was collected for NICS prior to embryo transfer. The clinical outcomes of patients implanted with euploid embryos were significantly better than those of patients implanted with aneuploidy embryos but did not differ from those of patients implanted with chaotic abnormal/NA embryos. NICS combined with morphological grading can be clinically used to select blastocysts for transfer in frozen-thawed cycles. Embryo suitability for transfer is in the order of ‘euploid embryo with good morphology’, ‘chaotic abnormal/NA embryo with good morphology’, ‘euploid embryo with fair morphology’, and ‘chaotic abnormal/NA embryo with fair morphology’(Table
3 and Additional file
2: Figure S2). Meanwhile, the clinical outcomes of patients were not related to the fertilization approach in the niPGT-A cycle, which might provide patients with a new treatment strategy where both the fertilization approach and PGT can be noninvasive.
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