Introduction
In vitro fertilization (IVF) has proved beneficial for assisted reproductive technologies by opening new possibilities for couples experiencing infertility. An important step in this process is the selection of embryos with the highest survival rate and the lowest risk of genetic abnormalities (1). Preimplantation genetic testing (PGT) has serves an important role in this selection, allowing the detection of chromosomal aneuploidies and single-gene disorders before embryo transfer (2). However, conventional PGT methods, such as blastomere, polar body and trophectoderm biopsy, rely on invasive biopsy procedures, which not only raise ethical concerns but may also create a small risk to embryo viability and implantation potential. In previous years, the identification of cell-free DNA (cfDNA) in embryo spent culture media has proven a novel perspective in non-invasive PGT (niPGT) (3). During early embryonic development, fragments of nuclear and mitochondrial DNA are released into the culture medium of the embryo, providing a key source of genetic information without exposing the embryo to invasive procedures. This discovery represents a promising method for both research and clinical applications, as it enables genetic screening while preserving the structural integrity of the embryo. By using advanced molecular techniques such as digital PCR and next-generation sequencing, niPGT aims to provide a comprehensive and reliable assessment of the genetic profile of the embryo, offering the potential to improve implantation outcomes, reduce the risk of heritable diseases and optimize reproductive success in IVF programs (4).
Rhesus D (RhD) incompatibility between the mother and the fetus occurs when an Rh-negative mother carries an Rh-positive fetus. In this context, maternal antibodies cross the placenta and destroy fetal RhD antigens (5). Without any treatment, this condition can result in fetal anemia, hydrops fetalis, neonatal jaundice (hyperbilirubinemia) and even fetal mortality. Rh-induced hemolytic disease of the fetus and newborn (HDFN) is a blood disorder that is triggered by the incompatibility between a mother and fetus; this disease remains an important clinical problem, with the worldwide prevalence decreasing from ~99 to 44 cases per 100,000 live births after the introduction of routine anti-D immunoprophylaxis, which is applied to prevent RhD incompatibility (6,7). In this approach, Rh-negative pregnant women receive anti-D immunoglobulin at ~28 weeks of gestation to reduce the risk of fetomaternal hemorrhage. The RhD antigen is considered one of the most important immunogenic antigens in transfusion medicine (8). RhD antigens are encoded by the RHD gene, which is located on chromosome 1, close to the Rh blood group CcEe antigens (RHCE) gene (Fig. 1). RHD and RHCE genes exhibit 92-97% sequence similarity and both genes contain 10 exons. This high similarity can make genetic testing more difficult due to the risk of cross-reaction (9).
In non-invasive RHD genotyping, exon 7 and exon 10 are the main targets. RHD exon 7 is important as it includes variations specific to RHD, while exon 10 is used as an additional control to improve accuracy and reliability. Using both exons together helps to separate RHD from RHCE, resulting in an advantageous method for non-invasive prenatal RHD genotyping. Non-invasive methods for fetal RhD determination also exhibit the potential to influence infertility treatments. In particular, cell-free DNA (cfDNA), found in embryo culture media during IVF, has emerged as a new biological source for PGT (4). These analyses are performed without harming the embryo and non-invasive preimplantation genetic testing has attracted great attention in recent years (4,10). Specifically, for RhD genotyping, this method provides the possibility to detect Rh incompatibility without physical intervention on the embryo (9).
An important application of IVF is the screening and prevention of sex-linked genetic disorders, which are inherited conditions caused by mutations on the X or Y chromosomes and often exhibit distinct patterns of inheritance and clinical manifestation between men and women (11). These disorders are influenced not only by the genome but also by lifestyle, nutrition, environment and sex hormones such as estrogen, progesterone and testosterone, which affect immune responses and gene expressions (12). The X chromosome carries ~867 genes that are not only involved in sex determination but also serve notable roles in the development and function of major organs such as the brain, heart, liver, kidneys and skin, as a number of these genes encode structural proteins, enzymes and regulatory molecules necessary for normal cellular and physiological processes. X-linked disorders are usually more severe in male patients as men carry only one X chromosome, while females may exhibit milder symptoms due to a second X chromosome being present. However, abnormal X chromosome inactivation or homozygosity can still cause serious disease in females, including >500 X-linked disorders, including Duchenne muscular dystrophy, hemophilia A and fragile X syndrome, which appear to be more severe in males (12).
Since the Y chromosome is present only in males, Y-linked disorders are inherited exclusively through the paternal line and their phenotypic expression is restricted to male patients, as females lack the Y chromosome. An important gene on the Y chromosome is sex-determining region Y (SRY), located at p11.2 (Fig. 2) (13). This gene produces the testis determining factor, which initiates testis development. During early development, SRY activates other genes such as Sox9, leading to Sertoli cell formation and anti-Müllerian hormone production, which directs male reproductive system formation. Mutations or deletions of the SRY gene can cause conditions such as gonadal dysgenesis or hypospadias. Therefore, SRY is a key biomarker for sex differentiation and genetic diagnosis (13).
As the mitochondrial function of reproductive cells deteriorates, the quality of the germ cells declines and in the majority of cases, it prevents healthy embryo development (14). Mitochondrially encoded cytochrome b (MT-CYB) makes it possible to check for the quality of the reproductive cells beforehand as a part of niPGT. MT-CYB encodes a key protein component of mitochondrial complex III of the respiratory chain (the cytochrome bc1 complex), which serves a central role in cellular energy production through oxidative phosphorylation. Mitochondrial complex III is located in the mitochondrial inner membrane (15) and is responsible for catalyzing the electron transfer from ubiquinol to cytochrome c (16). Thus, it represents an important step in establishing the proton gradient across the inner mitochondrial membrane (17). This complex also has 11 subunits, of which only one subunit is encoded by mitochondrial DNA and is named cytochrome b. MT-CYB gene is located between the positions 14747 and 15887 of mitochondrial DNA (Fig. 3) and the cytochrome b protein, which is the only protein subunit that is encoded by mitochondrial DNA, is 380 amino acids long (17). In addition, the relatively high abundance of this gene in mitochondrial genomes, as well as its key function in oxidative phosphorylation, make MT-CYB an ideal target for quantification studies such as droplet digital PCR (ddPCR). Notably, pathogenic variants in the gene have been associated with mitochondrial complex III deficiency, impaired cellular energy production, and a broad spectrum of clinical disorders affecting tissue viability and organismal health (18).
The sensitivity of MT-CYB expression and mutations to cellular stress and metabolic changes makes it a potential biomarker of mitochondrial dysfunction, especially in IVF (19). In previous years, it has become common to analyze cfDNA samples in niPGT. Recent advancements in niPGT have allowed the use of cfDNA and mitochondrial cfDNA (cf-mtDNA) samples to identify the qualities of examined germ cells. Subsequently, this increases the chance of obtaining pregnancy with advanced maternal age by allowing the monitoring of reproductive cell quality (20).
Therefore, the aim of the present study was to identify Rh incompatibility, which can be lethal for the embryo, by analyzing the Rh factor in cfDNA obtained from spent culture medium (SCM) through niPGT and ensuring correct embryo selection before implantation. In addition, it was intended to use the identified genes associated with sex-linked diseases for screening, thereby providing an effective method for selecting the healthiest blastocyst. Furthermore, the present study sought to investigate whether there is an association between the amount of cfDNA in SCM and maternal age, with the aim of providing insights for future research as a part of promising biomarkers in niPGT.
Discussion
niPGT has emerged as a new approach in reproductive medicine that avoids invasive techniques and reduces risks, such as structural damage to the embryo or mosaicism associated with biopsy. Since the first discovery of cell-free fetal DNA in maternal plasma in 1997(29), researchers have started to move towards less invasive methods for genetic analysis in assisted reproductive technologies. The identification of cfDNA and cf-mtDNA in SCM represented new possibilities for analyzing embryonic genetic status without compromising embryo viability. With the help of technological advancements such as ddPCR, these techniques established the way for highly sensitive and precise quantification of genetic material from minimal and fragmented DNA sources, offering marked clinical potential for embryo selection in IVF.
In the present study, cfDNA and cf-mtDNA isolated from SCM were analyzed using ddPCR to understand their potential in niPGT. The present findings showed that ddPCR offered a highly sensitive detection of low-input genetic targets, an important advantage in niPGT applications whereby cfDNA quantities are often limited and highly fragmented.
Despite ddPCR having demonstrated high analytical sensitivity for RHD detection in low-input samples, the clinical concordance rate remained moderate. The clinical data concordance rate was 60% for five samples, considering the challenging nature of SCM-derived cfDNA. This discrepancy may have partly stemmed from the intrinsic characteristics of SCM-derived cfDNA, which contain a variable mixture of embryonic and maternal DNA fragments. The present findings are clinically relevant given that accurate RhD genotyping is important in preventing HDFN. Previous studies have shown that maternal DNA contamination and allelic drop-out are major limiting factors in RhD genotyping when fetal or embryonic DNA input is low (29,30). In addition, the RHD gene exhibits structural complexity, including pseudogenes and hybrid alleles, which may lead to false-positive or false-negative results depending on the region being targeted (31). These biological and technical constraints may explain why exon 7 and exon 10 amplification results were not always consistent across samples. To reduce these diagnostic risks in future clinical applications, the present study recommends employing a more effective multiplexing approach. Targeting at least three exons of the RHD gene at the same time in a single ddPCR reaction (such as exons 5, 7 and 10) could make detection notably more precise, compared with just relying on one or two exons.
Despite these limitations, the fact that ddPCR was able to detect RHD in fragmented cfDNA samples highlights its robustness and supports its potential for future niPGT applications, particularly when combined with contamination-reduction strategies or embryo-specific molecular markers. Previous studies using qPCR have reported reduced sensitivity at low cfDNA concentrations and in early gestation stages (32). By contrast, ddPCR partitions the DNA template into thousands of nanoliter-sized droplets, allowing absolute quantification of target sequences without the need for standard curves, thus providing a higher tolerance to PCR inhibitors (33). This may explain why, in the present study, ddPCR could reliably detect RHD sequences even in samples with low cfDNA input, supporting its advantages compared with conventional PCR methods for niPGT.
SRY gene amplification was successful in 65.3% of samples, with 100% concordance in the five cases with available pregnancy outcome data. SRY detection exhibited a higher clinical concordance compared with RHD, which may be attributed to its male-specific nature and the absence of maternal background interference. However, the overall amplification rate may indicate that SRY detection is still challenged by the low abundance of Y-chromosome fragments in SCM. Studies have reported that Y-derived cfDNA is often less abundant due to both biological factors (for example, fewer copies of Y-chromosomal sequences) and technical factors such as preferential degradation of longer Y-chromosome segments (34,35). The 100% concordance in the clinically validated samples nevertheless suggested that when detectable, ddPCR provided highly reliable sex determination for niPGT. It also supports that ddPCR may improve the precision of fetal sex determination, particularly in pregnancies at risk of X-linked disorders, by detecting male-specific DNA at earlier gestational ages and with greater sensitivity compared with qPCR.
It has previously been determined that the condition of mitochondrial DNA serves a key role in understanding the quality of the embryo and that this quality decreases as the maternal age advances (20). In the present study, the quality of cf-mtDNA samples derived from SCM was identified and evaluated with the MT-CYB gene as a part of a potential biomarker in niPGT. Mitochondrial cfDNA analysis with MT-CYB gene droplet count using ddPCR revealed a positive correlation between maternal age and cf-mtDNA levels, suggesting that mitochondrial stress and dysfunction increase with advancing maternal age, ultimately impacting embryo quality. In a study by May-Panloup et al (36), it was stated that as the age of the mother progresses, accumulation of the mtDNA mutations in oocytes increases and not only deteriorates the quality of the oocyte but also increases the risk of allowing mitochondrial abnormalities inherited by the offspring (36).
Given the central role of mitochondria in embryo quality and development, this finding may indicate an age-associated increase in mitochondrial dysfunction in oocytes, which in turn could contribute to reduced fertility. An elevated cf-mtDNA/cf-nDNA ratio may reflect enhanced release of damaged or dysfunctional mitochondria into the extracellular space, potentially impairing the overall health and developmental competence of the oocyte, unless there is embryo fragmentation or degeneration. In the case of embryo fragmentation and degeneration, release of mt-DNA into the SCM may increase regardless of embryo quality and maternal age. Under optimal conditions, which state physiologically intact and healthy embryo samples, the cf-mtDNA/cf-nDNA ratio effect, as presented in Fig. 6, was consistent with the findings of Liu et al (37), further supporting the evidence that cf-mtDNA levels increase with maternal age. The cf-mtDNA/cf-nDNA ratio graph shown in Fig. 6 has also been reported in a study by Tsirka et al (38), where the ratio was investigated in the context of IVF treatment. In this study, follicular fluid samples were analyzed using qPCR with the ribulose-5-phosphate-3-epimerase gene for cf-nDNA detection. The study found a direct association between increasing maternal age and elevated cf-mtDNA/cf-nDNA ratios. Although the amplification methods and primers used in the present study differed from those employed by previous studies (37,38), the present results corroborate their findings. Nevertheless, this observation should be considered an exploratory finding due to the limited sample size (n=6) and thus requires validation in larger cohorts.
In recent studies, >100 different samples have been analyzed (37,38). Compared with these studies, the reliability of the present findings was statistically less powerful in terms of detecting significant correlations due to the smaller dataset. Thus, obtained results of the present hypothesis should be valued as preliminary and require larger sample data set analysis to eliminate the main limitation in the present research for further studies. The relatively low detection rate of β-globin (12/33 samples) suggested that nuclear cfDNA may be present at lower abundance or may be more susceptible to degradation compared with mitochondrial DNA in SCM. Therefore, apparent increases in mtDNA relative to β-globin should be interpreted cautiously, as they may reflect reduced recovery or detectability of nuclear cfDNA rather than true mitochondrial enrichment alone. Despite these limitations, the present research holds marked value as it was conducted within a Turkish patient group and provides a basis for future studies with larger datasets. Expanding this research within the Turkish patient population has the potential to introduce a notable advancement in reproductive medicine. Anticipating embryo quality prior to implantation may increase the likelihood of achieving successful pregnancies in couples undergoing IVF for certain indications. Consequently, IVF success rates and embryo selection strategies may be improved. Particularly in cases where age-associated declines in oocyte quality are anticipated, cf-mtDNA level measurements could guide the selection of the most viable embryos for implantation, thereby enabling successful pregnancies, particularly in women of advanced maternal age. Overall, the application of niPGT utilizing cf-mtDNA demonstrates potential clinical utility with minimal invasiveness.
The sample size in the present research was concordant with the G*Power analysis, however was below the threshold for the specificity and sensitivity. Due to the fragmented structure of the cfDNA samples, the amplification rate, despite being higher compared with qPCR, was not satisfactory in all targets. The reliability of ddPCR results when cfDNA is fragmented beyond amplification was evaluated by Liu (39), who demonstrated how some genes, although present, can be silent during the early embryonic stages. In the early stages of embryonic development, even housekeeping genes may preserve a heterochromatin structure, therefore are not part of transcription.
Furthermore, early-stage embryos may release a limited quantity of nuclear DNA and the presence of mitochondrial DNA-rich but nuclear DNA-poor droplets could lead to stochastic loss of the target genes during partitioning, such as housekeeping genes. This limitation may also explain the inconsistent amplification observed between different targets, particularly between RHD exon 7 and exon 10, whereby unequal representation of fragmented genomic regions may result in exon-specific drop-out. Therefore, negative amplification results should be interpreted cautiously, as they may reflect technical limitations associated with low-input fragmented cfDNA rather than the true absence of the target sequence. Genes with high embryo-specificity, such as SRY, demonstrate higher clinical concordance, whereas targets susceptible to maternal background noise or genomic structural variation, such as RHD, require additional optimization. Inclusion of additional RHD targets, such as multiplexing methods of at least three exons, may improve detection reliability and concordance in future studies. In addition, the incorporation of internal embryonic DNA markers or polymorphic markers may function as a quality control mechanism to determine the presence of adequate embryonic DNA, thereby differentiating a genuine Rh-negative result from a technical amplification failure. This indicates that although niPGT exhibits good potential for diagnosis based on biomarkers, in the present context, it may be insufficient to determine a precise conclusion and should instead be used as a predictive model before further analysis.
An additional limitation of the present study was the absence of systematic validation using paired conventional embryo biopsy-based PGT results. Since embryo sex determination is not permitted to be reported under ethical regulations in Turkey and RHD status is not routinely evaluated within standard PGT protocols (40), clinical data were not available for all embryos included in the present study. Therefore, outcome information could only be obtained from a limited number of transferred embryos that resulted in live birth, where postnatal sex and/or Rh status could be determined. Although these limited follow-up data showed general concordance with SCM-based findings, larger prospective studies incorporating paired biopsy-PGT validation and broader clinical follow-up are required to determine the diagnostic accuracy and clinical applicability of this non-invasive approach. In addition, the correlation between the cf-mtDNA/cfDNA ratio and maternal age warrants further investigation. The subsample used for this specific correlation consisted of only 6 samples (n=6), which was markedly below the minimum sample size established by the present G*Power analysis. Therefore, the findings for this endpoint are underpowered and should be considered strictly preliminary. Nevertheless, cf-mtDNA-based parameters may still serve as promising non-invasive biomarkers in future studies.