Introduction
Embryo quality is a key determinant of success in assisted reproductive technology. With advancements in embryo culture and cryopreservation, it has become preferable to culture embryos to the blastocyst stage, as blastocyst transfer typically results in improved pregnancy outcomes compared to cleavage-stage transfer, largely due to better synchronization between the embryo and the endometrium [1]. Typically, blastocyst development is expected to occur between 114 and 118 hours after insemination, commonly referred to as day 5 blastocysts [2]. However, some embryos exhibit delayed development, only reaching the blastocyst stage on day 6 or even day 7 [3]. The impact of this delayed progression to the blastocyst stage on pregnancy outcomes, in comparison with day 5 blastocysts, remains uncertain. A 2019 systematic review and meta-analysis demonstrated a significantly higher clinical pregnancy rate and live birth rate with day 5 blastocyst transfer versus day 6 transfer [4]. The debate continues regarding the clinical implications of transferring blastocysts on day 5 or day 6, as some studies report comparable outcomes, while others support a preference for day 5 transfers [5-8].
Beyond morphological assessment, preimplantation genetic testing for aneuploidy (PGT-A)—which selects embryos with a normal chromosomal complement—is believed to increase the likelihood of achieving pregnancy and live birth. However, current evidence does not support routine clinical use of PGT-A, as it has not been shown to improve live birth rates [9]. Additionally, ongoing debate surrounds the impact of PGT-A on pregnancy outcomes, especially when the timing of blastocyst biopsy varies. Recent studies indicate that transferring a euploid blastocyst on day 6 to 7 yields lower pregnancy outcomes than transfer on day 5 [10]. These findings suggest that embryo morphology in combination with PGT-A may be insufficient for accurately assessing embryo quality, due to the complex regulation of each chromosome by extensive DNA and RNA interactions. This is in line with recommendations from several organizations advocating for multidimensional strategies to enhance embryo selection [9,11].
Omics technologies are emerging tools for assessing oocyte and embryo quality by investigating interactions between cellular structures and processes. These approaches extend beyond ploidy status and morphological assessment, aiming to reveal intrinsic variations and offer a more comprehensive evaluation of embryo competence between day 5 and day 6 blastocysts [12-14]. Among these, transcriptomics, which involves analyzing which genes are transcribed and their expression levels over specific time periods, has gained traction compared to proteomics and metabolomics, which so far lack sufficient evidence for improving reproductive outcomes [15,16]. Transcriptomic analysis has been extensively applied to human blastocysts, potentially revealing gene expression differences linked to varying clinical outcomes [17-20]. High-throughput methods such as microarray and RNA sequencing have enabled examination of a broad array of gene expressions, leading to the identification of several novel genes whose functions are yet to be determined [21,22]. Unlike polymerase chain reaction (PCR), which assesses a limited set of gene expressions, these techniques allow for broader profiling of specified genes [23,24].
Implantation-related genes, such as those involved in adhesion molecules, extracellular matrix components, steroidogenesis, and chemokine signaling, are considered vital for embryo-endometrium communication and may play a crucial role in determining pregnancy outcomes [18,20,25]. Nevertheless, understanding of the specific genes involved in embryonic implantation remains limited and often controversial, particularly regarding differences between normal (day 5) and delayed (day 6) blastocyst development. To address whether delayed (day 6) blastocyst development influences clinical outcomes, our study compared the expression of implantation-related genes between euploid blastocysts developed on day 5 and those developed on day 6. In addition, we evaluated the association between the expression of implantation-related genes and pregnancy outcomes in both categories of euploid blastocysts.
Methods
1. Participant recruitment and ovarian stimulation protocol
Prior to the commencement of this study, approval was obtained from the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (Approval number 0764/65), and the study was registered with the Thai Clinical Trials Registry (Registration number TCTR20230711003). All participants provided explicit and voluntary consent to participate during the recruitment period from January 2023 to February 2024. Informed consent was specifically obtained from all infertile couples enrolled in the study.
Infertile couples were eligible if the female partner was aged 18 to 42 years and indicated for fertility treatment with intracytoplasmic sperm injection (ICSI) at the Reproductive Biology Unit, King Chulalongkorn Memorial Hospital, Thailand. All couples were required to comply with the PGT-A guidelines as established by Thai regulatory authorities. The criteria for PGT-A included: female age 35 years or older, a history of at least two pregnancy losses within the first 12 weeks of gestation, failure to achieve implantation after two consecutive embryo transfers, a previous child with a genetic abnormality preventable by PGT-A, or the need for human leukocyte antigen matching to treat a prior child’s condition. Exclusion criteria were as follows: body mass index (BMI) >30 kg/m², uterine pathology affecting pregnancy outcomes (such as fibroids, adenomyosis, endometrial polyps, or intrauterine adhesions), or ovarian pathology that could impact gene expression profiles (such as polycystic ovary syndrome or endometriotic cyst).
Ovarian stimulation was carried out using either follitropin-alfa (Gonal-f; Merck Serono) or follitropin beta (Puregon; Organon), both recombinant gonadotropins. The gonadotropin dose, ranging from 150 to 300 IU/day, was individualized according to patient age, ovarian reserve testing (including anti-Müllerian hormone and antral follicle count), and prior response to stimulation cycles. When dominant follicles reached 14 mm in diameter, a gonadotropin-releasing hormone (GnRH) antagonist, ganirelix (0.25 mg; Orgalutran, Organon), was administered to suppress luteinizing hormone activity. Final oocyte maturation was triggered with a GnRH agonist, triptorelin (0.2 mg, Decapeptyl; Ferring Pharmaceuticals), followed by transvaginal ultrasound-guided oocyte retrieval performed 36 to 36.5 hours later.
2. Embryo culture and trophectoderm biopsy
Cumulus oophorus complexes were denuded of surrounding cells using the hyaluronidase enzyme within 2 hours of oocyte retrieval. Oocytes at the metaphase II stage were then fertilized via ICSI. Resulting embryos were cultured in a continuous single culture medium (Global Total LP; CooperSurgical) in an incubator set to 37 °C, with 6% CO₂ and 5% O₂, until they reached the expanded blastocyst stage on either day 5 or day 6. Assisted hatching was performed on day 4 of embryo development using a laser technique to promote trophectoderm (TE) cell protrusion. The laser was set to 100% power with a pulse duration of 120 microseconds. TE biopsy was subsequently performed on blastocysts by applying a flicking motion against the holding pipette, obtaining 5 to 10 cells on either day 5 or day 6. Biopsied TE cells were divided into two portions after being loaded into a buffer-containing tube. One portion was used for PGT-A, performed using next-generation sequencing with the Illumina MiSeq sequencing system (Illumina). The other portion was reserved for gene expression analysis. Only blastocysts confirmed as euploid (46, XX or 46, XY) by PGT-A were subjected to further gene expression analysis. A maximum of two euploid blastocysts per participant were included in subsequent real-time quantitative PCR (RT-qPCR) analyses.
3. Relative gene expression analysis
Total RNA was extracted from biopsied TE cells using the PicoPure RNA Isolation Kit (Applied Biosystems), specifically designed for high-quality RNA extraction from samples containing fewer than 10 cells, following the manufacturer’s instructions. Briefly, TE cells were lysed in extraction buffer and incubated at 42 °C for 30 minutes. The supernatant containing extracted RNA was then purified using a spin column. Purified RNA underwent library construction for downstream reverse transcription. The total RNA concentration was quantified with a spectrophotometer (Thermo Scientific NanoDrop Spectrophotometers). Conversion of RNA to cDNA was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). A 2× RT master mix was prepared, and 10 μL of RNA sample was added to each 20 μL reaction. Samples were loaded into a thermocycler and subjected to an initial incubation at 25 °C for 10 minutes, followed by 37 °C for 120 minutes, and a final step at 85 °C for 5 minutes. The cDNA concentration was measured with a spectrophotometer, then diluted to 50 ng/μL with DNase-free water and stored at –80 °C until analysis.
cDNA samples were further diluted to 10 ng/μL and used as templates for PCR amplification of the following target genes: C-X-C chemokine receptor type 4 (CXCR4), C-X-C motif chemokine ligand 12 (CXCL12), 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1), 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1), integrin subunit alpha V (ITGAV), laminin subunit alpha 1 (LAMA1), mucin 15 (MUC15), and the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These genes are implicated in various stages of embryo implantation, including hatching, apposition, adhesion, and invasion (Figure 1). Primer sequences are shown in Table 1. RT-qPCR reactions were performed using the QuantStudio Real-Time PCR System (Applied Biosystems) and KAPA SYBR FAST qPCR Master Mix (2X) (Kapa Biosystems). Each 20 μL reaction contained 2 μL of diluted cDNA and 10 μM of each forward and reverse primer. Cycling conditions followed the manufacturer’s instructions, with annealing temperatures ranging from 54 to 60 °C, depending on the primer set, for a total of 45 cycles. Each sample was analyzed in duplicate, in two separate runs, with negative controls (nuclease-free water) included. Relative quantification was determined using the comparative cycle threshold (Ct) method, with GAPDH as the reference gene. The relative expression of each target gene was calculated using the 2-ΔΔCT method.
4. Embryo transfer and pregnancy outcomes
A single embryo transfer was performed in a frozen cycle, prepared by administering a fixed dosage of 6 mg per day of estradiol valerate (Progynova; Bayer AG) starting on the third day of the menstrual cycle. After a minimum of 12 days of exogenous estrogen administration, transvaginal ultrasound was used to assess endometrial thickness. When the endometrium reached at least 8 mm with a triple-layer appearance, patients began micronized progesterone vaginal suppositories (Utrogestan; Besins Healthcare). The transfer of a single euploid embryo was carried out, with selection based on morphological grading according to the 2011 Istanbul consensus system. Embryo implantation was confirmed by measuring serum beta human chorionic gonadotropin levels equal to or greater than 5 mIU/mL on the 14th day after embryo transfer. Clinical pregnancy was defined by the presence of an intrauterine gestational sac detected by ultrasound 4 weeks after embryo transfer. Pregnancies were monitored through the 12th week of gestation to document the occurrence of spontaneous miscarriage.
5. Sample size calculation
A pilot study was undertaken to determine the required sample size by evaluating CXCR4 gene expression in four euploid blastocysts from each group. The mean±standard deviation (SD) relative expression of CXCR4, expressed as fold change, was 0.0523±0.0683 for day 5 euploid blastocysts and 0.4019±0.3856 for day 6 euploid blastocysts. Based on these results, with a statistical power of 0.80 and an alpha level of 0.05, the calculated sample size required was 10 euploid blastocysts per group.
6. Statistical analysis
Statistical analysis was performed using SPSS ver. 28.0 (IBM Corp.). Raw expression data, represented by Ct values, were normalized using GAPDH Ct values as the housekeeping gene. Fold changes in gene expression between day 5 and day 6 blastocysts were calculated using the 2-ΔΔCt method. Continuous data were presented as mean±SD and compared using the unpaired t-test. Non-parametric data were reported as median and interquartile range and analyzed using the Mann-Whitney U test. Categorical data were expressed as numbers (%) and analyzed with Fisher’s exact test. A p-value <0.05 was considered to indicate statistical significance.
Results
1. Participants and baseline characteristics
Thirty-two infertile women were screened for study eligibility. Fifteen women were found to have euploid blastocysts suitable for transcriptomic analysis. Among these participants, three had euploid blastocysts available on both day 5 and day 6. As shown in Table 2, there were no statistically significant differences in baseline characteristics, including age, partner’s age, BMI, and ovarian reserve testing, between participants in the day 5 and day 6 groups. The primary factors contributing to infertility were unexplained causes and male factor infertility. All participants were nulliparous and had no prior history of ovarian stimulation.
2. Embryo characteristics and pregnancy outcomes
Twenty-three day 5 blastocysts and 19 day 6 blastocysts underwent PGT-A. Of these, 20 blastocysts were confirmed as euploid: 10 from day 5 and ten from day 6. Analysis of embryo morphology and quality revealed no statistically significant differences in grading or chromosomal results between euploid blastocysts from days 5 and 6 (Table 3). Among transferred embryos, 60% of day 5 euploid blastocysts and 75% of day 6 euploid blastocysts resulted in ongoing pregnancies, with no cases of spontaneous miscarriage observed in either group.
3. Comparison of gene expression levels between day 5 and day 6 euploid blastocysts
Relative gene expression levels are summarized in Table 4. CXCR4 expression was low in day 5 blastocysts, but showed a significant increase in day 6 blastocysts (p=0.031) (Figure 2). CXCL12, HSD17B1, ITGAV, and LAMA1 were upregulated and exhibited high expression in both day 5 and day 6 blastocysts, with no significant differences between groups. In contrast, HSD3B1 and MUC15 were downregulated in both groups (Figure 3). Notably, 35% of samples for HSD3B1 and ITGAV expression had indeterminate Ct values. Analysis of euploid blastocysts obtained on both days from the same participants revealed no significant differences in gene expression for the examined genes.
4. Relative gene expression according to pregnancy outcomes
Ten embryos from day 5 euploid blastocysts and four embryos from day 6 euploid blastocysts were transferred via a single embryo transfer protocol. Nine transfers of euploid blastocysts led to successful clinical pregnancies, corresponding to a clinical pregnancy rate of 60% for the day 5 group and 75% for the day 6 group. Comparison of relative gene expression between pregnant and non-pregnant groups showed no statistically significant differences (Table 5). However, subgroup analysis of day 5 euploid blastocysts (Table 6) demonstrated that CXCR4 expression was significantly lower in the pregnant group compared to the non-pregnant group (Figure 4), while there were no significant differences in the expression levels of the remaining six target genes.
Discussion
In this study, we used RT-qPCR to evaluate the expression levels of seven genes in TE cells related to embryonic implantation. A comparison was made between euploid blastocysts showing normal development on day 5 and those exhibiting delayed development on day 6. Overall, our results demonstrated differential expression of implantation-related genes in euploid blastocysts on days 5 and 6. Notably, CXCR4 expression was significantly higher and upregulated in day 6 euploid blastocysts compared to day 5 blastocysts, which showed downregulation. Furthermore, among day 5 euploid blastocysts, CXCR4 expression differed substantially between those that achieved successful implantation and those that did not. While no statistically significant differences were found in the expression of CXCL12, HSD17B1, ITGAV, and LAMA1 between day 5 and day 6 euploid blastocysts, all of these genes showed upregulation. By contrast, HSD3B1 and MUC15 were consistently downregulated in both groups.
1. Relative CXCR4 expression levels in euploid blastocysts between day 5 and day 6
Currently, there are no quantitative comparisons in the literature regarding CXCR4 expression between euploid blastocysts on day 5 and day 6. Our findings revealed that relative CXCR4 expression in day 6 euploid blastocysts was significantly higher than in those from day 5. Specifically, CXCR4 was upregulated in day 6 blastocysts, while it remained downregulated in day 5 blastocysts. This suggests that CXCR4 expression in euploid embryos fluctuates dynamically during embryonic development, reaching peak levels prior to implantation. Since day 6 blastocysts are typically more developmentally advanced than day 5 blastocysts, they may exhibit increased CXCR4 expression, particularly in the TE lineage preparing for implantation. This is consistent with previous work by Bao et al. [19], who reported that CXCR4 expression first appeared at the 4-cell stage, increased at the 8-cell stage, and peaked at the blastocyst stage. Unlike Bao’s study [19], our data showed that CXCR4 expression in day 5 euploid blastocysts remained low. This discrepancy may be due to Bao’s inclusion of integrated blastocyst-stage embryos, without differentiation between days 5 and 6, when assessing expression. Furthermore, all embryos in our study were of high quality and underwent PGT-A to confirm chromosomal status, ensuring their suitability for transfer.
CXCR4 functions as a chemokine receptor within the G protein-coupled receptor family and is involved in regulating the proliferation and differentiation of several cell types, including those in human blastocysts [26,27]. It also plays key roles in migration and invasion, which are processes crucial for implantation, by facilitating interactions between the trophoblast cells of the blastocyst and the endometrium [28,29]. This embryo-endometrium crosstalk is further supported by the detection of CXCR4 expression in the endometrium, particularly during the implantation window, where it enhances endometrial receptivity through modulation of angiogenic markers, integrins, or leukemia inhibitory factor [30-33]. Dominguez et al. [29] reported that CXCR4 expression in the endometrium is upregulated briefly during the mid-luteal phase, corresponding with the window of embryo implantation. This aligns with our observation of increased CXCR4 expression in TE samples from day 6 blastocysts. Simultaneous upregulation of CXCR4 in both day 6 blastocysts and the endometrium during the implantation window may enhance maternal–fetal communication, thereby increasing the probability of successful pregnancy.
2. Relative CXCR4 mRNA expression compared between euploid blastocysts in pregnant and non-pregnant participants
No previous studies have systematically examined CXCR4 expression between pregnant and non-pregnant groups after single euploid blastocyst transfer on days 5 and 6. In our pooled analysis of all euploid blastocysts, there were no differences in the expression of target genes between pregnant and non-pregnant groups. However, subgroup analysis of day 5 euploid blastocysts revealed a substantial difference in CXCR4 expression: blastocysts from pregnant individuals had lower CXCR4 expression than those from non-pregnant individuals. The former displayed downregulation, while the latter showed slight upregulation. We hypothesize that aberrant CXCR4 expression in day 5 euploid blastocysts, specifically early upregulation, may negatively impact implantation. This is supported by previous findings that aberrant CXCR4 expression can induce apoptosis in TE cells and impair their migration and adhesion to the endometrium [18].
Limited data are available for day 6 euploid blastocyst transfers, as only four such embryos were included in our study, and no prior research has addressed this comparison. Considering the observed trend of increasing CXCR4 expression in day 6 euploid blastocysts and the expression differences seen in the day 5 embryo transfer subgroup, as well as previous work [19], it could be speculated that, in day 6 embryo transfers, CXCR4 expression may be higher in the pregnant group than in the non-pregnant group, potentially achieving a significant expression difference. Nevertheless, caution is warranted when interpreting the results reported by Bao et al. [19], as their embryos were from donor women who subsequently became pregnant, rather than from the same embryos that produced pregnancies. In addition, the transfer of varying numbers of embryos in fresh cycles is unusual in this clinical context. In contrast, our study used high-quality, PGT-A-confirmed embryos that were actually transferred in a frozen cycle with a single embryo transfer, reflecting real-world clinical practice.
3. Comparison of other implantation-related genes between day 5 and day 6 euploid blastocysts
Although there were no statistically significant differences, the expression levels of CXCL12, HSD17B1, ITGAV, and LAMA1 were upregulated, whereas HSD3B1 and MUC15 were downregulated, regardless of the day of blastocyst development or pregnancy outcome. Each of these genes serves distinct roles during implantation.
CXCL12, a ligand specific to CXCR4, is secreted by the embryo and induces CXCR4 expression in the endometrium [34]. This underscores the importance of CXCR4/CXCL12 expression in embryos throughout implantation. In our study, CXCL12 expression was upregulated, but no differences were observed between day 5 and day 6 blastocysts, which contrasts with the pattern seen for CXCR4.
LAMA1 encodes laminin alpha 1, which participates in attachment, adhesion, migration, and cell signaling during implantation [35]. Laminin also serves as a ligand for integrin. In this study, we analyzed the αv subunit encoded by ITGAV, one of the integrin subunits, and found upregulated expression of both LAMA1 and ITGAV in TE cells. Furthermore, no difference in their expression was found between pregnant and non-pregnant groups. In contrast, Kirkegaard et al. [18] reported higher LAMA1 expression in non-implanting blastocysts. This discrepancy may be explained by differences in methodology, as the previous study used RNA sequencing without RT-qPCR validation, which may have limited the accuracy of quantitative measurements.
The steroid metabolism-regulating genes HSD3B1 and HSD17B1 exhibited contrasting expression patterns, with HSD17B1 being upregulated and HSD3B1 downregulated. Previous studies have indicated that both genes are upregulated in TE cells [17,35]. Additionally, HSD17B1 expression was found to be downregulated in non-competent embryos [20], and it has been hypothesized that downregulation of HSD17B1 expression would negatively impact the process of implantation, as indicated by its correlation with repeated miscarriages [36]. Our results showed comparable HSD17B1 mRNA expression levels irrespective of blastocyst day or pregnancy outcome. Unlike earlier reports, we found HSD3B1 expression to be downregulated in both groups, with approximately 35% of samples yielding indeterminate Ct values. HSD3B1 expression varies among human trophoblast subtypes, being higher in extravillous trophoblasts than in cytotrophoblasts [37]. In summary, TE cells exhibited a limited level of expression until they differentiated into trophoblasts.
MUC15 is a membrane-bound mucin glycoprotein, and its function in TE remains poorly understood [17]. Most research on MUC15 focuses on trophoblast and placental tissue. Overexpression of MUC15 suppresses trophoblast-like cell invasion by promoting tissue inhibitor of metalloprotease production, which counters matrix metalloproteinase activity. Therefore, low MUC15 expression promotes trophoblast invasion [38]. Our results showed consistently low MUC15 expression on both day 5 and day 6 euploid blastocysts, making comparative analysis difficult due to the extremely low expression levels.
4. Strengths and limitations
The main strength of this study is its prospective design, focusing exclusively on good-quality blastocyst-stage embryos with confirmed normal chromosomal status by PGT-A, all of which were transferred individually in frozen cycles. This approach differs from earlier studies that used retrospective designs, donor oocytes or embryos, poor-quality blastocysts, or embryos with unknown chromosomal status. However, our study also has limitations. We were unable to confirm protein expression for each gene studied. The limited number of day 6 euploid blastocyst transfers also prevented comparison of gene expression with pregnancy outcomes in this group. Additionally, we could not determine a definitive threshold to distinguish CXCR4 expression levels between pregnant and non-pregnant groups.
In conclusion, we compared gene expression profiles related to implantation in euploid blastocysts on days 5 and 6, as well as between implanted and non-implanted euploid blastocysts. A key finding was the significant upregulation of CXCR4 in day 6 euploid blastocysts compared to day 5, and the observation of lower CXCR4 expression in successfully implanted day 5 blastocysts compared to those that did not implant. No statistically significant differences were observed in the expression of other implantation-related genes, including CXCL12, HSD3B1, HSD17B1, ITGAV, LAMA1, and MUC15. We hypothesize that CXCR4 expression increases over time until the implantation period, and that dysregulation of CXCR4 expression at specific stages of blastocyst development may impair implantation.
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