Introduction
Globally, at least 10 to 13 million infants have been born through assisted reproductive technology (ART) in the 40 years since the first successful birth in 1978 [1]. The live birth rate per initiated ART cycle is approximately 20% to 30%, indicating a failure rate of 70% to 80% for a single attempt [2,3]. This issue is particularly salient for patients of advanced maternal age, among whom the likelihood of embryo aneuploidy is substantially elevated [4,5]. For couples of advanced age, determining the ploidy status of the embryo selected for transfer can improve treatment success rates [6,7].
Preimplantation genetic testing for aneuploidy (PGT-A) via next-generation sequencing (NGS) combined with trophectoderm (TE) biopsy of blastocysts has been increasingly employed to select embryos with normal ploidy for transfer [8,9]. However, while PGT-A increases the likelihood of live birth and reduces recurrent miscarriages, it is an invasive and costly procedure that can deter some patients. Furthermore, it is not universally available due to varying regulatory limitations across countries [10]. The invasive nature of this method also raises questions about potential effects on the subsequent development of biopsied embryos. Concerns also include negative impacts on clinical outcomes, increased obstetric or neonatal risks, and unknown health risks to offspring [11,12]. Consequently, demand is increasing for non-invasive embryo selection methods in ART that can identify embryos with the highest potential for successful pregnancy.
One widely used non-invasive embryo selection method, the blastocyst grading system—developed by Gardner and Schoolcraft—is based on morphological parameters such as expansion grade, inner cell mass (ICM), and TE [13]. Research has indicated that blastocysts with high-grade morphology are more likely to be euploid, with consistent evidence of a significant relationship between blastocyst morphology and ploidy [14]. When morphology is considered alongside PGT-A, higher-grade blastocysts are more likely to be euploid, whereas those with lower morphology and expansion grades are more likely to be aneuploid [15]. In assessing blastocyst euploidy, prediction improves when both developmental speed and the day of blastocyst formation are considered. Developmental speed appears to be a particularly effective predictor of ploidy, comparable to key morphological parameters. A previous study reported that evaluating blastocyst developmental speed—measured from insemination to blastocyst expansion—better predicts ongoing pregnancy and delivery rates [16]. However, several studies have found that day 5 euploid blastocysts have higher implantation and live birth rates than similarly graded day 6 blastocysts [17]. Therefore, determining morphological parameters and developmental speed constitutes an effective, non-invasive approach for selecting blastocysts for transfer. Recent studies have proposed additional non-invasive methods to predict euploidy, including time-lapse technology and non-invasive PGT-A using culture media [18-21]. Although promising, these technologies are still under development and impose high financial costs on patients [20,22]. The most predictive parameters for blastocyst ploidy and sex remain unclear, particularly regarding developmental speed.
For patients who face challenges with blastocyst selection from frozen embryos or who lack access to advanced technologies such as PGT-A, selection relies solely on morphological parameters. This study aims to clarify how the morphological parameters and developmental speed of blastocysts may correlate with ploidy and sex, providing insights to improve embryo selection for in vitro fertilization (IVF). Our findings could facilitate the effective selection of euploid blastocysts using morphology and developmental speed, thereby increasing IVF success rates.
Methods
1. Patients and study design
This retrospective cohort analysis included 856 blastocysts from 439 patients who underwent PGT-A at the Ojinmed IVF Center, Mongolia, between May 2019 and June 2023. All patients received clomiphene citrate (CC) according to a minimal ovarian stimulation protocol or natural cycle protocol devised by Kato et al. [23,24]. The CC (Fuji Pharma) regimen was 50 mg/day from the third day of the menstrual cycle until final oocyte maturation. If needed, follicle-stimulating hormone (Gonal-f; Denk Pharma) was added at a total dose of 300 to 600 IU. Ovulation triggering was performed with a nasal spray containing the gonadotropin-releasing hormone agonist buserelin. Oocyte retrieval was usually performed 35 hours after triggering, without anesthesia, using a 21- or 22-G needle (Kitazato). Follicular flushing was conducted using a solution of modified human tubal fluid (HTF) with HEPES (Kitazato) and 1% heparin when the leading follicle was 18 mm or larger.
2. Embryo culture, blastocyst monitoring, and vitrification
Cumulus‒oocyte complexes were collected and transferred to HTF (Kitazato) with 10% serum protein substitute (SPS; Kitazato) for insemination at 37 °C under 5% O2, 6% CO2, and 90% N2. Before insemination, cumulus cells surrounding the oocytes were removed, and the oocytes were transferred to HTF medium supplemented with paraffin oil (Kitazato). Insemination via intracytoplasmic sperm injection (ICSI) using micropipettes (TPC Micropipettes; CooperSurgical) was carried out 4 to 5 hours after retrieval. The sperm source comprised only ejaculated sperm processed with density gradient centrifugation. Until insemination, sperm were cultured in HTF with phenol red (Kitazato) supplemented with SPS. After insemination, oocytes were individually transferred to 30-µL drops of one-step medium (Naka IVF medium; Naka Medical) at 37 °C under 5% O2, 6% CO2, and 90% N2 in water-jacketed or dry incubators (APC-50DR and EC-6S; Astec), and the time of insemination was recorded on the patient card. At 16 to 20 hours after ICSI, zygotes with two pronuclei were cultured to the blastocyst stage. Per our IVF center’s protocol, day 5 to day 6 embryos were closely monitored two to four times daily. Observations typically began at 8:00 AM; if an embryo had reached the blastocyst stage, its size was checked every 3 hours until 5:00 PM. If it had not yet reached the blastocyst stage, it was checked again 6 hours later at 2:00 PM and then 3 hours later at 5:00 PM. If embryos still had not reached the blastocyst stage—with an inner diameter of at least 160 µm or at the hatching stage—they were checked again the next day at 8:00 AM. Blastocyst assessments were performed only by well-trained senior embryologists, and each observation was completed within 1 minute. The time to blastocyst formation was defined as the number of hours post-insemination (hpi) at which the embryo first reached the blastocyst stage, occurring on either day 5 or day 6 of development. The time to blastocyst expansion was defined as the hpi at which the blastocyst first met the following criterion: a fully formed cavity with a diameter ≥160 µm. This developmental stage was recorded upon its first observation on day 5 or day 6.
3. Embryo culture and TE biopsy
Fully expanded blastocysts with inner diameters of at least 160 µm (corresponding to expansion grade ≥3) had a hole drilled in the zona pellucida (ZP) using a laser (wavelength, 1.48 μm infrared diode; duration, 1.8–2.2 ms; Octax NaviLase; Vitrolife). ZP drilling was performed using an inverted microscope (Eclipse TE-2000U; Nikon), and blastocysts were shrunk with a high-osmolality solution (Dilution Solution; Kitazato) [25]. Two to four hours later, five to six TE cells were harvested from each blastocyst. Biopsied TE cells were washed four to five times with phosphate-buffered saline (Kitazato) and transferred to a polymerase chain reaction tube containing 4.5 µL of buffer solution. Samples were stored at −20 °C until analysis. After biopsy, blastocysts were vitrified using the Cryotop method (Kitazato), as described previously [26].
4. Whole-genome amplification and NGS
Whole-genome amplification was performed using the ChromInst Kit (Yikon Genomics) according to the manufacturer’s instructions. The DNA concentration of the purified library mixture was determined with a Qubit 2.0 fluorometer (Thermo Fisher Scientific) following the manufacturer’s protocol. To analyze the ploidy status of the biopsied TE samples, libraries were sequenced on a MiniSeq sequencer (Illumina). This sequencing enabled detection of whole-chromosome aneuploidies, mosaicism, and segmental aneuploidies (deletions or duplications greater than 4 Mb). Embryos were classified as mosaics if 20% to 80% of cells were abnormal. Samples with less than 20% abnormal cells were classified as euploid, whereas those with more than 80% were considered aneuploid. The copy number variation sequencing (CNV-seq) platform, previously validated for mosaicism detection via cell mixing experiments, was used for these analyses.
5. Statistical analysis
All data analyses were performed using SPSS ver. 15.0 (SPSS Inc.). Continuous data are presented as means and standard deviations. Nominal variables were analyzed with the Fisher exact test, and associations between two categorical variables were assessed using the chi-square test for trend, as appropriate. Differences between groups for continuous variables were assessed by analysis of variance. p-values less than 0.05 were considered to indicate statistical significance. Multivariable logistic regression was performed and adjusted for paternal age, baseline hormone levels, and body mass index (BMI). Results are reported as adjusted odds ratios (aORs) with 95% confidence intervals (CIs).
6. Ethics approval and consent to participate
This study was approved by the Ethical Committee of Ojinmed IVF Center (Approval number: EA2024022703). All participant data were obtained with informed consent. All procedures performed in studies involving human participants were in accordance with the ethical standards of the 1964 Helsinki Declaration and its later amendments.
Results
This retrospective cohort study included 856 blastocyst PGT-A results from 619 oocyte pick-up cycles involving 439 patients who underwent clomiphene-based minimal stimulation. The average maternal age was 35.87±5.67 years, and the average paternal age was 36.5±6.4 years. The total number of oocyte pick-up cycles was 619, with a mean of 1.92±1.67 cycles per patient. A total of 3,392 oocytes were retrieved, of which 2,619 (77.2%) were mature (metaphase II). Additional information on causes of infertility, BMI, baseline hormone levels (estrogen, thyroid-stimulating hormone, prolactin, follicle-stimulating hormone, and anti-Müllerian hormone), blastocyst formation and expansion rates, and general patient characteristics is shown in Table 1.
The correlations of blastocyst ploidy percentage and sex with factors such as maternal age, paternal age, and morphological parameters (including ICM, TE, day of blastocyst, formation time, and expansion time) are shown in Table 2. A total of 972 blastocysts were biopsied, and 856 were analyzed via NGS. Of these, 426 (49.8%) were euploid, 286 (33.4%) were aneuploid, and 144 (16.8%) were mosaic. Euploidy, aneuploidy, and mosaicism rates were significantly correlated with maternal age (with mean ages of 34.69±4.5, 38.19±3.88, and 35.63±4.75 years, respectively; p<0.001) but not with paternal age (39.01±6.45, 39.71±6.58, and 37.8±4.76 years, respectively; p>0.05). Additionally, abnormalities were significantly correlated with maternal age (whole aneuploidy, 38.57±3.6; segmental aneuploidy, 35.67±4.55; p<0.001) but not with paternal age (whole aneuploidy, 39.01±6.96; segmental aneuploidy, 39.79±6.57; p>0.05). The chi-square test revealed that various blastocyst morphological parameters—including ICM, TE, day of blastocyst, blastocyst formation time, and expansion time—were significantly associated with ploidy rate. Significant differences in euploidy, aneuploidy, and mosaicism rates were observed across ICM and TE grades, although whole aneuploidy and segmental aneuploidy rates did not differ significantly. Day of blastocyst development, formation time, and expansion time were correlated with ploidy: shorter development times were associated with higher euploidy and lower aneuploidy rates. Blastocyst sex was correlated only with blastocyst vitrification time (male, 127.9±10.25 hpi; female, 129.68±10.8 hpi; p=0.02) (Table 2).
Multivariable logistic regression was conducted to evaluate the contributions of maternal age, ICM grade, TE grade, day of blastocyst, formation time, and expansion time as predictors of chromosomal abnormalities. Compared with women younger than 35 years, women 35 years or older had 3.34-fold greater odds of aneuploidy (aOR, 3.34; 95% CI, 2.35 to 4.61; p<0.001) and 71% lower odds of euploidy (aOR, 0.29; 95% CI, 0.21 to 0.41; p<0.001). Maternal age was not correlated with mosaicism or blastocyst sex. Among the morphological parameters, only TE grade was significantly correlated with euploidy and aneuploidy rates. Compared with TE grade A, TE grade C was associated with 3.06-fold greater odds of aneuploidy (aOR, 3.06; 95% CI, 1.42 to 6.10; p<0.01) and 68% lower odds of euploidy (aOR, 0.32; 95% CI, 0.15 to 0.67; p<0.01). Other morphological parameters, including ICM, day of blastocyst, formation time, and expansion time, were not significantly correlated with ploidy rates. Blastocyst sex was correlated only with expansion time; each additional hour of expansion was associated with a 5% decrease in the likelihood of a male blastocyst (aOR, 0.95; 95% CI, 0.91 to 0.99; p=0.03). Blastocyst sex was not correlated with other blastocyst morphological parameters or parental age (Table 3).
Discussion
The higher euploidy rates observed in younger women may be linked to a reduced likelihood of meiotic errors during oocyte maturation [27]. As women age, chromosome segregation errors become more common, increasing the risk of aneuploidy—which is strongly associated with maternal age—and elevating the risk of chromosomal disorders in offspring [4]. Maternal age is a key factor in embryo selection protocols, as older women may benefit from additional genetic screening or adjustments to stimulation protocols to improve outcomes. Santamonkunrot et al. [28] reported euploidy rates of 52.7% in women aged 35–38 years, 34.7% in those aged 38–40 years, and 18.4% in women older than 40 years. Similarly, Kim et al. [15] reported that maternal age had a major effect on embryo euploidy. Nair et al. [29] described an overall aneuploidy frequency of 46.8% in 15,112 day 5 blastocysts. The incidence was approximately 28% in women under 30 years and increased steadily to about 67% in women over 40 years; whole-chromosome aneuploidy rose from 27.8% to 67.5% across the same age groups. Most studies have found that paternal age has no significant effect on chromosomal abnormality rates. Consistent with this, our data showed that paternal age was not significantly correlated with blastocyst ploidy. However, women aged ≥35 years, compared with those <35 years, displayed increased aneuploidy and decreased euploidy rates. The mosaic rate was not correlated with maternal age. Additionally, maternal age and morphological parameters were associated with both euploidy and whole-chromosomal abnormalities. Previously, TE grade has been reported to be only slightly more correlated with segmental chromosomal abnormalities than other parameters [30]. In our study, both whole and segmental abnormalities were significantly associated with maternal age, with women younger than 35 years having lower percentages of both.
Previous studies have reported that higher ICM and TE grades and faster expansion rates are strongly correlated with blastocyst ploidy [15,31]. Capalbo et al. [14] found that blastocysts with the highest morphological scores had higher euploidy rates (excellent, 56.4%; good, 39.1%; average, 42.8%; poor, 25.5%). They also observed that complex aneuploidy involving multiple chromosome errors was significantly associated with poorer morphology (6.8%, 15.2%, 17.4%, and 27.5% for excellent, good, average, and poor quality, respectively) [14]. Wang et al. [32] examined 1,159 blastocysts and reported significant correlations between TE grade, expansion grade, and euploidy rate. In our study, TE grade showed a stronger correlation with euploidy than did developmental speed and ICM grade. This association is biologically plausible given the role of the TE in hatching and implantation. Moreover, human chorionic gonadotropin secreted by TE cells exerts immunomodulatory effects that are essential for establishing a successful pregnancy [33]. In multivariable analysis, among morphological parameters, TE grade C was significantly associated with a greater likelihood of aneuploidy.
In addition, delayed developmental speed has been observed more often in aneuploid than in euploid blastocysts, with delays in the onset of compaction, time to morula, time to blastulation, and time to reach the blastocyst stage [18,34,35]. Lou et al. [36] reported that the euploidy rate of blastocysts decreases when development is delayed to day 7, with euploidy rates on days 5, 6, and 7 of 49.5%, 36.5%, and 32.9%, respectively. Moreover, Huang et al. [37] found that male embryos developed faster than female embryos, particularly at the three-, four-, and five-cell stages. Multivariate logistic regression showed that blastocyst sex was significantly correlated only at the three-cell stage (OR, 0.786; 95% CI, 0.62 to 0.98; p<0.05) [37]. Wang et al. [38] reported that higher grade and more rapid development in the blastocyst group were associated with a greater likelihood of male birth compared with slow development and poor-grade blastocyst transfer (aOR, 0.46; 95% CI, 0.23 to 0.92; p<0.05). In bovine blastocysts, embryos that reached the blastocyst stage earlier were predominantly male, with male blastocysts exhibiting better morphology and higher hatching rates than female blastocysts [39]. Notably, high-quality TE may contribute to more efficient ion pumping, which can increase fluid accumulation in the blastocoel cavity. Expansion of the blastocoel cavity—a critical aspect of blastocyst development—is influenced by the number of cells forming the blastocyst, particularly TE cells; increased cell numbers result in a larger cavity, which can facilitate faster hatching [40,41]. Our results corroborate that male blastocysts expand more rapidly at the blastocyst stage, whereas blastocyst sex was not correlated with other morphological parameters.
In conclusion, this study underscores the significant impact of advanced maternal age on blastocyst aneuploidy rates, with women under 35 years exhibiting lower rates of chromosomal abnormalities. Among morphological parameters, TE grade was significantly correlated with euploidy rate. Blastocyst sex was correlated with time to blastocyst expansion, with male blastocysts expanding more quickly than female blastocysts. This study is the first to report correlations between blastocyst morphological parameters, chromosomal abnormalities, and blastocyst sex. These insights advance the understanding of how maternal age and blastocyst parameters can predict chromosomal abnormalities and sex, aiding in the refinement of embryo selection criteria in ART.
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