Optimization of Detecting Embryonic cell free DNA in Spent Blastocyst Media: A Non-invasive Approach for Preimplantation Genetic Screening

Lama Khudari¹*, Marwan Halabi², Sahar Al Fahoum¹

¹Lama Khudari, Dept. of Biochemistry and Microbiology,

Faculty of Pharmacy, Damascus University, Damascus, Syrian Arab Republic.

²Marwan Halabi, Dept. of Anatomy, Histology and Embryology, Faculty of Medicine, Damascus University, Damascus, Syrian Arab Republic.

³Sahar Al Fahoum, Dept. of Biochemistry and Microbiology, Faculty of Pharmacy, Damascus University, Damascus, Syrian Arab Republic.

*Corresponding Author E-mail: lama.khudari@damascusuniversity.edu.sy

ABSTRACT:

Background: Preimplantation genetic screening (PGS) is essential in assisted reproductive technology (ART), enabling couples to assess embryos for genetic abnormalities before implantation during in vitro fertilization (IVF). This study investigates and validates a protocol for isolating cell-free fetal DNA (cffDNA) from spent blastocyst media, positioning it as a non-invasive biomarker for evaluating embryo chromosomal integrity. Our research aims to deepen the understanding of embryonic health and viability, which could lead to improved reproductive outcomes. By focusing on the use of cffDNA in spent culture media, this study addresses a significant gap in reproductive medicine, offering new insights into embryo assessment and potentially enhancing the selection process for viable embryos. Methods: This is a methodological cross-sectional study. We selected 21 women under 43 years old and collected 27 SBM samples from blastocyst-stage embryos. We tested various collection variables, including SCM volume, timing (Day 3 vs. Day 5), and storage conditions (fresh vs. frozen). We refined our cffDNA isolation technique and validated it via 24-chromosome aneuploidy screening using Next Generation Sequencing (NGS) on five SCM samples. Results: Our study successfully extracted free embryonic DNA (cffDNA) from media collected on day 5 of blastocyst culture, particularly utilizing fresh spent blastocyst medium (SBM) samples. We identified that the optimal volume for these SBM samples is less than 20 µl, with concentration values ranging from 2.59 to 56 ng/µl. Additionally, we found that the most effective method for isolating cffDNA from SBM is DOP-PCR, which is well-suited for amplifying the entire gDNA. Gel electrophoresis revealed amplification products as smears ranging from 200 bp to 2000 bp, indicating successful amplification. Regarding aneuploidy screening for the five SBM samples, our optimized protocol proved effective. We identified three euploid samples, categorized as normal female (XX), alongside one aneuploid sample (7:1,18:1, X0). One additional sample displayed a noisy NGS profile. Conclusion: Our results emphasize the reliability of our protocol for extracting cffDNA from spent Blastocyst Medium (SBM) and assessing the genetic status of the embryos, providing valuable insights for future research in reproductive genetics.

KEYWORDS: Spent Blastocyst Media, Non-Invasive PGT-A, Embryonic cell free DNA, Next generation Sequencing (NGS), Preimplantation genetic screening (PGS).

INTRODUCTION:

During the early stages of embryo development, chromosomal abnormalities known as aneuploidies can lead to problems such as growth arrest, repeated implantation failure, and recurrent miscarriage^1,2. These aneuploidies are significant factors that impact the success rates of In vitro fertilization (IVF)^3,4,5. They may arise from meiotic errors, often linked to increased maternal age⁶, or post-zygotic errors after fertilization. Notably, even embryos from fertile couples can be aneuploid, with higher rates observed in IVF-conceived embryos compared to natural conception⁷.

Preimplantation Genetic Testing (PGT) is effective but raises embryo safety concerns and requires standardized procedures. Noninvasive Preimplantation Genetic Testing (niPGT) offers a promising alternative, reducing costs and enhancing outcomes. Research indicates that niPGT reliably detects genetic anomalies with lower error risks compared to invasive methods, utilizing noninvasive monitoring of spent culture media in 2016⁸, minimizing embryo manipulation and biopsy risks⁹.

Trophectoderm biopsy, performed on blastocysts at days 5 to 7 after oocyte retrieval, collects 5 to 10 cells for genetic testing. While this method is less harmful to implantation than earlier-stage biopsies, it remains invasive and requires skilled personnel and specialized equipment. Removing cells can impact implantation success, and concerns over genetic misdiagnosis highlight the limitations of embryo biopsies, often regarded as the gold standard. This has sparked interest in developing non-invasive approaches to Preimplantation Genetic Testing for Aneuploidy (PGT-A) to minimize risks while offering valuable genetic information¹⁰.

The detection of cell-free DNA (cfDNA) in spent culture media (SCM) from human embryos suggests its potential as a biomarker for embryo assessment. This insight enables non-invasive Preimplantation Genetic Testing (niPGT), a safer alternative to traditional biopsy. Utilizing cfDNA from SCM or blastocoel fluid, niPGT shows promise for efficient genetic evaluation of preimplantation embryos from IVF. However, further research is needed to confirm its reliability for clinical applications¹¹.

Variable success rates and discrepancies between noninvasive Preimplantation Genetic Testing for Aneuploidy (niPGTA) and trophectoderm biopsy can result from factors like DNA quality, embryonic mosaicism¹², and analysis methods, leading to uncertainty about the clinical efficacy of niPGTA^13,14.

In this study, we optimize and validate a protocol for isolating embryonic cell-free DNA (cfDNA) from spent culture media after blastocyst development. We aim to assess whether the extracted cfDNA accurately reflects the chromosomal composition of the blastocyst, enhancing both quantitative and qualitative analysis of chromosomal status. We investigate whether cfDNA from spent culture media can reliably indicate the genetic status of the embryo, improving insights into embryonic health and viability.

MATERIALS AND METHODS:

Patients and study sample:

This methodological Cross-Sectional Prospective Study, conducted from March 2022 to May 2023 with ethical approval from the University of Damascus Ethics Committee (No. 2/A.K. dated 3/1/2020), involved 21 women under 43, with a mean age of 34.2 years (SD = 5.1). Participants were selected based on criteria including low pregnancy rates, previous unsuccessful IVF attempts, recurrent miscarriages, a family history of hereditary diseases, or a desire for gender determination. All women received treatment at Al-Sharq Hospital for Assisted Fertility and provided written informed consent after being fully informed about the study's purpose and procedures.

A total of 27 spent culture medium (SCM) samples were collected from blastocyst-stage embryos, which underwent morphological evaluation by a fertility specialist according to criteria from the Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. Only high-quality embryos, classified as Grade 1 with fragmentation rates not exceeding 15%, were included.

After harvesting the spent culture medium, 27 trophectoderm biopsies (TE) were taken on days 3 or day 5 of incubation for preimplantation genetic testing (PGT) to determine the sex of the embryos, as requested by parents. Two discarded embryos¹⁵ were used as a positive control (PC), while a control sample of culture medium without any embryo served as a negative control (NC). Contamination was minimized by careful handling and washing procedures for each embryo to ensure the integrity of the samples.

Optimization conditions:

We optimized the collection of spent culture media from blastocysts by testing graduated volumes ranging from 75 µl down to 10-20 µl. This approach aimed to determine the ideal volume for effective extraction, ensuring that we have both sufficient quantity and quality of cell-free fetal DNA (cffDNA) for molecular diagnostic tests typically conducted on trophectoderm biopsies.

The optimal time of embryo development (Day 3 vs Day 5) was also evaluated to yield sufficient cell-free fetal DNA (cffDNA). Additionally, we explored various storage conditions for the spent culture medium, including fresh samples, those stored at +4°C, and those frozen at -20°C and -80°C. This helped us establish the ideal conditions for sample collection to maximize cffDNA yield.

Isolation of Cell-free DNA:

Embryonic cell free DNA (cffDNA) was isolated and amplified from the Spent Blastocyst Media (SBM) using the DOPlify® WGA kit (PerkenElmer) according to the manufacturer’s instructions, and the Whole Genome Amplification (WGA) to increase the yield of fetal DNA and subsequently purify it. This method relies on polymerase chain reaction using degenerate oligonucleotide primed PCR (DOP-PCR) that are suitable for amplifying the entire gDNA in vitro to obtain a sufficient quantity for analysis of low copy number gDNA (<100pg). This is the amount that is usually present when isolating DNA from a single cell.

Quantification of Cell-free DNA:

The concentration of cffDNA was quantitatively analyzed using a Qubit™ 4 Fluorometer with a Qubit dsDNA HS Assay kit. Then, we verified that the DNA sample was amplified and there was no contamination in the negative control sample by performing electrophoresis on an agarose gel at a concentration of 1% and in the presence of a DNA gradient with a range extending from 100 to 3000 base pairs. Electrophoresis was performed by applying 2 µl of PCR product, for 30 minutes at a current of 100 volts. The DNA products from the whole genome amplification appear in the form of a smear ranging from 200bp to 2000bp.

Protocol validation:

The custom protocol was validated to obtain good quantities and qualities of cffDNA and adopted for preimplantation genetic screening for aneuploidy (PGT-A). This meticulous approach aimed to enhance the accuracy and reliability of the genetic testing process. For this purpose, the cell-free fetal DNA (cffDNA) isolated from spent blastocyst media was assessed with 24-chromosome aneuploidy screening by Next Generation Sequencing (NGS) uses Nextera technology (Illumina) to provide accurate screening of all 24 chromosomes (1-22, X and Y) for the identification and selection of euploid embryos and determine whether the DNA yield is sufficient for Preimplantation genetic testing for aneuploidy (PGT-A). This advanced approach offers a comprehensive evaluation of genetic information for improved accuracy and reliability in embryo selection.

Statistical analysis:

We clustered our study’s samples (n=27) to 3 clusters to evaluate our protocol statistically. Group A (n=9) include freezing/ stored samples with Day3 and sample volume more than 50 µl, Group B (n=9) Fresh samples with Day 3/5 and sample volume 20-50 µl/ <20 µl, Group C (n=9) Fresh samples with Day 5 and sample volume less than 20 µl. Statistical analyses were performed using IBM SPSS Statistics Software version 24.

In our study, we aimed to compare categorical data. Given that our sample size was less than 30, we first assessed the normality of the data using the Kolmogorov-Smirnov test. We defined statistical significance as a P-value of less than 0.05. The results indicated that the data did not follow a normal distribution, prompting us to use a nonparametric test for our analysis. Therefore, we opted for the Kruskal-Wallis test (Table 1) to compare the groups effectively with P value less than 0.05 which means that there were differences between clusters that were considered to be statistically significant.

Table 1: Kruskal-Wallis test comparing groups A,B,C with P Value <0.05 which means that is statistically significant.

Test Statistics^a,b
	Sample Volume	Day3/Day5	Fresh/Freezing
Chi-Square	10.151	14.857	26.000
df	2	2	2
Asymp. Sig.	.006	.001	.000

a. Kruskal Wallis Test

b. Grouping Variable: Grouping

Informativity rates (Successfully samples/ Total analyzed samples) were estimated for these clusters and the informative results (Table 2) confirmed the optimal results for group C with informativity rate more than 66%.

RESULT:

This study evaluated the presence of cell-free fetal DNA (cffDNA) in spent blastocyst media (SBM) collected on days 3 and 5 of embryo development (n=27), comparing it with conventional trophectoderm biopsy for non-invasive preimplantation genetic testing (niPGT). We faced significant challenges isolating cffDNA from media on day 3 (n=5), with no meaningful amplification success or embryonic DNA yield. However, by day 5 (n=22), we successfully obtained free embryonic DNA by modifying our sampling and handling protocols. The best results came from fresh SBM samples processed immediately after collection (n=15). Attempts to preserve samples at +4ºC, -20ºC, or -80ºC proved ineffective for consistent DNA preservation (n=12). Additionally, we assessed the optimal volume of SBM by testing three ranges: >50µl (n=5), 20-50µl (n=5), and <20µl (n=17). Our results demonstrated that the highest yield of cffDNA came from samples less than 20µl, average concentration 14.1ng/µl, with a range of 2.59 to 56ng/µl. In contrast, concentrations for >50µl and 20-50µl samples averaged 4.432ng/µl and 1.98ng/µl, respectively (Diagram1).

Diagram1: The optimum volume of spent blastocyst culture medium samples was tested in the ranges of >50 µl (n=5), 20-50 µl (n=5), and <20 µl (n=17). It was found that the best SBM sample size to obtain a good yield of free embryonic DNA was <20 µl, where the average concentration of free embryonic DNA (cffDNA) after applying the isolation protocol and whole genome amplification (WGA) was 4.432 ng/µl for samples taken with volumes of >50 µl, and 1.98 ng/µl for samples taken with volumes of 20-50 µl, while the average concentration for samples with volumes of less than 20 µl was about 14.1 ng/µl (with a range of 2.59-56 ng/µl).

We evaluated the quality of cell-free fetal DNA (cffDNA) from spent blastocyst culture medium (SBM) samples by performing electrophoresis on agarose gels using products from whole genome amplification (WGA) across 27 SBM samples. cffDNA was identified in five samples (group C), characterized by a smear ranging from 200 bp to 2000 bp, with concentrations between 3.16 to 56ng/µl, all from fresh Day 5 samples under 20 µl volume. Notably, similar characteristics were observed in one sample from a discarded embryo. In contrast, the remaining 22 SBM samples from groups A and B showed no visible smear, indicating no detectable cffDNA (see Figures 1 and 2). Negative controls (no-template controls, NTC) also confirmed the absence of contamination. This analysis underscores the potential of recovering cffDNA from SBM for non-invasive genetic assessment.

Figure 1: Agarose gel electrophoresis illustrating the results of whole-genome amplification (WGA) of DNA from spent blastocyst culture medium (SBM) samples, as evidenced by distinct smears.

· Lane 1: DNA ladder ranging from 250 to 10,000 bp, used as a reference for size estimation.

· Lane 2: Negative control (no-template control, NTC), which is completely clean and free of contamination, confirming the integrity of the experimental conditions.

· Lane 3: A smear representing free embryonic DNA isolated from SBM medium, with sizes ranging from 300 to 1000 bp.

· Lanes 4-5: No amplification products observed, indicating the absence of detectable free embryonic DNA in these samples.

· Lane 6: Another smear of free embryonic DNA isolated from SBM medium, showing a range of 220 to 1200 bp.

· Lane 7: A sample from a discarded embryo, serving as a positive control for method validation,displaying a range of 250 to 3000 bp.

Figure 2: Agarose gel electrophoresis illustrating the results of whole-genome amplification (WGA) of DNA from spent blastocyst culture medium (SBM) samples, as evidenced by distinct smears. DNA ladder ranging from 250 to 10,000 bp.

· NC: Negative control, which is completely clean and free of contamination.

· Lane 1: A smear representing free embryonic DNA isolated from SBM medium, with sizes ranging from 200-600 bp.

· Lane 2: A smear representing free embryonic DNA isolated from SBM medium, with sizes ranging from 200-650 bp.

· Lane 3: A smear representing free embryonic DNA isolated from SBM medium, with sizes ranging from 200 to 750 bp.

· PC: Positive Control derived from a discarded embryo, displayed fragment sizes ranging from 250 to 3000 bp.

· Lane 4: No amplification products observed, indicating the absence of detectable free embryonic DNA in these samples.

A comprehensive 24-chromosome screening was performed using next-generation sequencing (NGS) with Nextera technology (PGT-A) with a MiSeq PGS (Illumina) on cell-free fetal DNA isolated from five spent blastocyst media (SBM) samples and one discarded embryo. This study screened all 24 chromosomes (1-22, X, and Y) by examining copy number variation (CNV) values, where a CNV of 1.2 or lower indicated complete loss, and values of 2.8 or higher indicated complete gain. Values between 2.0 and 2.4, and 2.4 to 2.8 were considered normal.

Results revealed that three SBM samples were euploid and confirmed as normal female (XX) (Figure 3), while one sample exhibited aneuploidy with monosomies of chromosomes 7, 17, and X (Figure 4). One sample showed a noisy NGS profile (Figure 5). Interestingly, the discarded embryo also displayed a normal female chromosomal profile. In a concordance assessment, we compared Fluorescence In Situ Hybridization (FISH) results from trophectoderm biopsy with our NGS findings. We observed good concordance in three samples with normal female (XX) results. However, one sample showed partial concordance with FISH indicating X0, and another sample produced a noisy amplification profile despite FISH indicating female (XX). This study enhances understanding of chromosomal integrity during early embryonic development.

Figure 3: Normal Female (Euploidy)- NGS

Figure 4: 7:1, 18:1, X0 (Aneuploidy)- NGS

Figure 5: Noise Amplification- NGS

DISCUSSION:

In our study, we meticulously programmed the harvesting of media from embryos cultured under controlled conditions, focusing on standardized culture medium volume, collection timing, and preservation methods. This optimization aimed to maximize both the quantity and quality of cell-free fetal DNA (cffDNA). As a result, we successfully isolated fragmented cffDNA released into the spent blastocyst medium (SBM).

On Day 5 of the embryo culture, our observations revealed significant presence of cffDNA in the SBM. The good quantities and quality of these DNA fragments correlate with the increasing number of cell divisions¹⁶ occurring as the embryos develop into the blastocyst stage. Notably, this cffDNA supports non-invasive preimplantation genetic testing (niPGT), allowing for genetic analysis without compromising embryo integrity. Our findings align with those reported by Yang and colleagues in 2017, further validating the feasibility of utilizing cffDNA from SBM for genetic testing¹⁷.

However, we did encounter issues with some samples during electrophoresis¹⁸, where no visible smears of amplified cffDNA were detected. This absence could result from low concentrations of isolated DNA or ineffective amplification due to the poor quality of fragmented DNA, undermining its suitability for genetic testing. The biological variability inherent in the media used and challenges in consistently collecting complete SBM droplets also contributed to reduced quantities of cffDNA in tested samples.

To enhance cffDNA concentration, we recommend minimizing the volume of spent embryo culture medium collected while ensuring complete harvesting. This approach would not only maximize cffDNA concentration in individual samples but also improve amplification and detection rates, thereby reducing variability across samples. These recommendations are reinforced by previous studies, including work by Vera-Rodriguez et al. (2018), Feichtinger et al. (2017), and Ho et al. (2018)^19,20,21.

Furthermore, we found that using degenerate oligonucleotide primers (DOP-PCR) for isolating and amplifying cffDNA was a highly effective method for whole genome amplification (WGA). This technique enabled us to achieve significant yields of embryonic DNA from blastocyst culture media, typically characterized by low genomic DNA concentrations. Importantly, this method adheres to our established conditions for preimplantation genetic testing (PGT), ensuring embryos remain intact throughout the process.

The detection technology chosen for quantifying cffDNA substantially influences the results as highlighted by Li et al. (2018)²². We employed fluorescent dye-based methods, such as the Qubit device, which specifically measures double-stranded DNA. This contrasts with spectrophotometric methods that evaluate all nucleic acids, including RNA, as demonstrated in the research by Shamonki et al. (2016) ²³ and Vera-Rodriguez et al. (2018)¹⁹, underscoring the importance of selecting appropriate detection technology for accurate cffDNA quantification.

Our hypothesis posits that DNA in the spent media represents the entire embryo, offering significant advantages for niPGT over traditional trophectoderm (TE) biopsy methods. TE biopsies may fail to provide a comprehensive representation of the entire embryo (Kuznyetsov et al., 2018; McCoy, 2017)^24,25. The debate surrounding the ability to isolate whole genomes from fragmented DNA in spent media necessitated further exploration of optimal conditions for genomic DNA collection. This exploration is critical for developing viable alternatives to current preimplantation genetic testing methods.

To validate our protocol, we analyzed cffDNA samples isolated from the spent medium, confirming our ability to extract high-quality, sufficient quantities of whole genomic embryonic DNA suitable for genetic screening. Our preimplantation genetic testing for aneuploidy (PGT-A) using next-generation sequencing (NGS) demonstrated the reliable isolation of whole genome cffDNA, reflecting genetic information for all 24 chromosomes. Samples with low DNA yields (NGS Noisy profile) were excluded, ensuring only viable embryos progressed for potential implantation. Analyzing the copy number variation (CNV) values, we assessed each embryo's genetic profile to classify them as euploid or aneuploid. This information is critical for making informed decisions in the context of preimplantation genetic testing.

Notably, our findings revealed strong concordance between sex determination results from TE biopsy and those obtained through SBM preimplantation genetic testing. This supports the effectiveness of our non-invasive approach, emphasizing its potential as a reliable alternative to traditional methods and optimizing embryo selection for successful pregnancies.

CONCLUSION:

Our study confirms the validity of using spent culture medium (SCM) as a source of cffDNA for detecting embryo aneuploidy noninvasively via NGS. Despite a small sample size, we successfully isolated high-quality cffDNA, highlighting SCM's potential for preimplantation genetic testing (PGT). This noninvasive approach offers an excellent alternative to invasive trophectoderm biopsies, providing a valuable option for couples at risk of genetic disorders in their offspring.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to express their appreciation to First Genomix Labs in UAE for their generous provision of full PGT-A services and for their cooperation throughout our research study. Great thanks to Dr. Samer Farawati for his valuable scientific advice, which greatly contributed to the success of this research.

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*TE_biopsy SBM Crosstabulation**
Grouping	TE_biopsy	DNA_TE		DNA_0	DNA_SBM	Total
A	TE_biopsy	DNA_TE	Count	9		9
	TE_biopsy	DNA_TE	% within TE_biopsy	100.0%	.00	100.0%
	Total		Count	9		9
	Total		% within TE_biopsy	100.0%	.00	100.0%
B	TE_biopsy	DNA_TE	Count	8	1	9
	TE_biopsy	DNA_TE	% within TE_biopsy	88.9%	11.1%	100.0%
	Total		Count	8	1	9
	Total		% within TE_biopsy	88.9%	11.1%	100.0%
C	TE_biopsy	DNA_TE	Count	3	6	9
	TE_biopsy	DNA_TE	% within TE_biopsy	33.3%	66.7%	100.0%
	Total		Count	3	6	9
	Total		% within TE_biopsy	33.3%	66.7%	100.0%