Mitochondrial DNA and genomic DNA ratio in embryo culture medium is not a reliable predictor for in vitro fertilization outcome

To investigate the ratio of mitochondrial DNA to genomic DNA (mt/gDNA) in embryo culture medium as a possible predictor for embryonic development and pregnancy outcome, we collected a total of 93 embryo biopsy specimens from 52 women at the corresponding Day 3 (D3) and Day 5 (D5) embryo culture medium of in vitro fertilization. With the multiple annealing and looping-based amplification cycles method of next-generation sequencing for whole genome amplification, we examined the karyotype of the biopsy samples and the mt/gDNA ratio in the culture medium. Results showed that the ratio of mt/gDNA had an upward trend with decreasing trophectoderm levels with no significant difference. At the same time, from D3 to D5, the mt/gDNA ratio in the medium of embryos that failed to become blastocysts showed an upward trend, and the mt/gDNA ratio of medium from embryos that reached blastulation with successful pregnancy showed a decreasing trend, but the differences were not statistically significant. We conclude that there is a certain correlation between mt/gDNA ratio and early embryonic development, but it does not reach a level that can be used as a clinical predictor.

www.nature.com/scientificreports www.nature.com/scientificreports/ millions of mtDNA copies in a single trophoblast cell 17,18 . From blastocyst fluid and embryonic body DNA was released into the culture medium that can be extracted for measurement [19][20][21] . Ravichandran et al. suggested that the mtDNA/gDNA ratio in the embryo culture medium was related to embryonic development, and it could be used, instead of biopsied embryo cells, to predict the quality and the outcome of embryo for implantation 22   D3 and D5 were experimental groups, and D3-NC1 (n = 10) and D5-NC1 (n = 10) were the control groups that identically processed but did not contain embryo, NC2 (n = 10) is the unused culture medium. Data are means + SEM. ***P < 0.005.

Figure 2.
Corresponding mt/gDNA ratio in embryo culture medium when embryonic euploidy is different. At D3, the mt/gDNA ratio corresponding to different euploidy in the embryo culture medium (a). Mt/gDNA ratio corresponding to different euploidy in D5 embryo culture medium (b). We divided the samples into euploidy and aneuploidy based on chromosome ploidy of the embryo biopsy samples, as shown on the abscissa. The ordinate represents the ratio of mt/gDNA in the culture medium. One black dot or black triangle represents one sample. Data are means ± SEM. (2019) 9:5378 | https://doi.org/10.1038/s41598-019-41801-1 www.nature.com/scientificreports www.nature.com/scientificreports/ ratio of embryo culture medium was related to blastulation and was expected to have a predictive value, while Victor et al. believed that mtDNA content had no correlation with blastocyst ploidy, age or viability [23][24][25] . This controversy has not been solved up to now. Therefore we performed a study to address this controversy.

Results
the experimental group and the control group. We designed two kinds of controls, one was named NC1 that was culture medium underwent the corresponding culturing process but did not contain embryos, and the other was named NC2 that was not used for culturing. The average numbers of mt/gDNA ratios of the D3 group and the D5 group were 158.38 (SEM = 18.39) and 206.24 (SEM = 30.24), respectively, and there was no statistical difference (P = 0.09). The average numbers of mt/gDNA ratios of the D3-NC1 group, the D5-NC1 group and the NC2 group were 30.3 (SEM = 12.38), 34.2 (SEM = 12.27) and 31.10 (SEM = 7.64), respectively. The experimental results showed that the mt/gDNA ratio in the experimental groups (D3 and D5) are much higher than that of the corresponding control groups (D3-NC1 and D5-NC1) (P < 0.001). At the same time, there was also a significant difference between the experimental group and NC2 (P < 0.001), while there was no significant difference between NC2 and NC1 (D3-NC1 and D5-NC1) (Fig. 1). embryonic chromosome euploidy and mt/gDNA ratio. To investigate the relationship between mt/ gDNA ratio and embryo chromosomal ploidy in culture medium, we compared the mt/gDNA ratio of the D3 and D5 embryos in 93 embryos and chromosome ploidy of the corresponding biopsy samples. The embryo is usually biopsied at D5 if the embryo was developed into the Grade 4 blastocyst or later stage. If on D5 the embryo had not matured to the Grade 4 blastocyst, we continued to culture it to D6 and then performed the biopsy. The embryos after biopsy were vitrified. The number of eggs biopsied can be found in Supplement Table 1. The results showed that 30 (32.2%) embryonic biopsy samples had a normal chromosomal euploidy, and 63 cases (67.7%) had uneuploidy. In D3 culture medium, the average numbers of mt/gDNA ratios of the euploid group and the aneuploid group were 78.95 (SEM = 35.19) and 124.9 (SEM = 20.57), respectively, and there was no statistical difference (P = 0.087). In D5 culture medium, the average numbers of mt/gDNA ratios of the aneuploid group and the abnormal group were 37.52 (SEM = 33.40) and 107 (SEM = 25.45), respectively, and there was no statistical difference neither (P = 0.21) (Fig. 2).

Figure 3.
Corresponding mt/gDNA ratio in embryo culture medium when the score of ICM is different. At D3, the mt/gDNA ratio corresponding to different score of ICM in the embryo culture medium (a). Mt/gDNA ratio corresponding to different score of ICM in D5 embryo culture medium (b). According to the scores of ICM in each blastocyst (A-C), the samples were divided into three groups, A, B, and C, such as the abscissa. The ordinate represents the ratio of mt/gDNA in the culture medium. One black dot, black triangle or black square represents one sample. Data are means ± SEM. www.nature.com/scientificreports www.nature.com/scientificreports/ Blastocyst quality and mt/gDNA ratio.  Age of women and mt/gDNA ratio. According to age, the patients were divided into three groups of Relationship between mt/gDNA ratio and pregnancy outcome. A total of 12 of the 52 cases were transplanted, and one embryo was transplanted in each case (Table S1). And the rest were frozen embryos that had were not transplanted. The 28-day B-ultrasound gestational sac and fetal heart rate were the criteria for success. Of the transplanted cases, 6 were successful and 6 failed. The results showed that on D3, the average numbers of mt/gDNA corresponding to pregnancy success and  www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
In this study, culture medium of the embryo on D3 and D5 before freezing (transplantation) was collected, and the mt/gDNA in the culture medium was detected using NGS to examine changes of released DNA during the culture process. We believe that the observed low baseline levels of DNA were derived from protein supplements originally contained in the culture medium. The main protein supplement was human serum albumin, which had a primary binding affinity of DNA 26,27 . When the commercial medium and protein supplements were analyzed separately, the highest level of DNA was found in 100% serum replacement supplement 28 . In our study, there was no significant difference in mt/gDNA ratio between NC1 and NC2, indicating that human operation and culture environment did not significantly affect the DNA content in the culture medium. Results showed that there was no significant correlation between euploidy and mt/gDNA of the embryo, but the average number of D3 and D5 in the normal group was lower than that in the abnormal group. There was a certain association between the two, but mt/gDNA could not be used as a clinical indicator to predict the ploidy of embryonic chromosomes as the changes were not statistically significant.
Based on our results, the ratio of mt/gDNA in D3 and D5 was increased slightly with the decrease of embryonic ICM score, but there was no statistical difference. Similarly, as the TE score decreased, the mt/gDNA in D3 and D5 also showed a small increase, and there was no statistical difference either. In addition, the proportion of fragments contained in the blastocyst did not significantly affect the mt/gDNA ratio in the medium (D3 and D5). Therefore, it appeared that mt/gDNA was related to the quality of embryos to a certain extent, but it could not be used as a clinical indicator in predicting the formation of capsules and superior embryos.
From D3 to D5, the mt/gDNA of embryos with successful pregnancy showed a downward trend, while the trend of non-pregnancy showed an upward trend, which was consistent with the trend of forming blastocysts. However, the changes did not reach the level of statistical difference. It could be speculated that mt/gDNA might have a certian correlation with embryo quality, but it could not be used as a clinical indicator to predict pregnancy outcome.
In recent years, the role of mitochondria in embryonic growth and development has attracted wide attention. There are different options on whether mitochondria can be used as a predictor of embryo quality. Some believed that mitochondrial DNA copy number in culture medium could be used as a non-invasive predictive index 23,24 . However, others believed that mtDNA content had no correlation with blastocyst ploidy, age or viability 25 . The results of our study, however, are inclined to show a relationship between mitochondrial DNA and embryo quality Figure 6. Mt/gDNA ratio in embryo culture medium corresponding to different outcome of blastulation. At D3, the mt/gDNA ratio corresponding to different outcome of blastulation in the embryo culture medium (a). Mt/gDNA ratio corresponding to different outcome of blastulation in D5 embryo culture medium (b). According to whether embryos grow to the blastocyst stage, the samples were divided into two groups, Yes and No, such as the abscissa. The ordinate of the ordinate represents the ratio of mt/gDNA in the culture solution. One black dot or black square represents one sample. Data are means ± SEM. www.nature.com/scientificreports www.nature.com/scientificreports/ and development, but the relationship was not significant enough to be used as an indicator to judge the quality of the embryos.
The conclusion of our study is different from those of the previous reports for several reasons: (1) Most of the studies that support mtDNA as a clinical predictor were based on the recognition of the "silent embryo hypothesis". This hypothesis was derived from early research on cattles and believed that there was a "mitochondrial genetic bottleneck" [29][30][31] . The copy number of mtDNA is significantly increased during oogenesis 12,32 and is actively reduced during pre-implant development 29 . The mtDNA sequence variants segregate between generations despite the high mtDNA copy number in the oocyte 23 . This led to the concept of "mitochondrial genetic bottleneck", which limits the process by which mtDNA molecules travel from the mother to the fetus and the offspring. Subsequent studies suggested that the copy number of mtDNA was significantly increased during egg production in large mammals 12,32-36 and then rapidly decreased during the pre-implantation developed phase 31,37 . However, a similar study on mice reached a different conclusion 33,38,39 . It appeared that similar studies of different species yielded different results. (2) At the same time, the mechanism of the mitochondrial genetic bottleneck mentioned above is still controversial. Some studies suggested that mitosis of mtDNA occured before primordial germ cell expansion to gonadal colonization 40 . On the other hand, other studies suggested that mtDNA bottlenecks occurred in the reduction of mtDNA copy number during development before implantation 39 . We collected the culture medium on D3 and D5 of early embryo development, and the results only reflected the changes of this stage. (3) Concerning target genes and detection technology, previous studies selected D-loop gene or other short fragments in mitochondrial DNA as the detection target to represent the copy number of mitochondrial DNA 35,41 . Studies showed that environmental stress might affect the transient synthesis of mitochondria after fertilization 42 . The D-loop region is more susceptible to variation due to environmental stress than other regions of mitochondrial DNA 43 . At the same time, previous studies used qPCR combined with specific probes to detect the D-loop region of mitochondrial DNA 35 , which increased the possibility of unstable results. Another study chose Alu, a multiple copies gene, as a detection fragment 44 , which was not technically superior to that of a single copy gene, and the frequency and composition of the Alu sequence are significantly different in population 45 . multiple annealing and looping-based amplification cycles (MALBAC) and NGS has been successfully used in various studies of DNA at single cell level, such as detecting single nucleotide variation (SNV) and copy number variation (CNV), analyzing meiotic recombination 46,47 , and performing PGS of in-vitro fertilized embryos 47,48 . Studies have also shown that NGS is sensitive enough to detect small differences between subculture populations from the same cell line and that MALBAC-NGS is also suitable to study mtDNA copy number in single cells with good reproducibility 49 . Most of the samples used in previous studies were from multiple clinics [22][23][24]28,50 , and the samples used in this study were from a single clinic. Factors such as media, culture temperature, biopsy techniques, and equipment used during sample collection would affect the outcome. (4) There were other contradictions www.nature.com/scientificreports www.nature.com/scientificreports/ among previous reports. In some studies samples were collected from both the cleavage and/or the blastocyst stages 28,50 , while other studies collected samples from the cleavage stage only 23 . The above may provide some but not all explanations for the discrepancy.
We conclude that the number of mitochondrial DNA copies released from embryos into the culture medium is related to embryo quality and development during in vitro culture, but it would not be a reliable clinical indicator to evaluate or forecast embryo development.

Methods patients selection.
We recruited 52 couples (median maternal age 32.7 years) from Jinjiang Women and Children's Hospital, Chengdu, China., who gave written informed consent. All couples underwent intracytoplasmic sperm injection (ICSI). This prevented paternal contamination from sperm and limited the maternal contamination from cumulus cells. Ethical approval was obtained from the Scientific and Ethical Committee of Jinjiang Women and Children Hospital for the experimental protocol (2018 KY-001). All clinical procedures including ova collection, in vitro fertilization, embryo culture and implantation were performed according to the standard hospital protocol (CDXN/QD-EMBYO-02-17) that has been approved by the Ethical Committee of Jinjiang Hospital which was accredited by ISO9001 and JCI (Joint Commission on Accreditation of Healthcare Organizations). Patient consent was obtained for each embryo sample used in this study.
Gamete preparation and ICsI procedure. Oocyte preparation. After the follicle became mature, human chorionic gonadotropin (HCG) was injected. Thirty-four to 36 hours later transvaginal ultrasound-mediated oocyte retrieval was performed. The ova were repeatedly pipetted in GMOPs Sperm preparation. We use the upstream method for semen optimization. Detailed steps are as follows: Figure 8. Mt/gDNA ratio in embryo culture medium corresponding to different age of woman (regardless of euploidy). At D3, the mt/gDNA ratio in the embryo culture medium corresponding to different age of woman (a). Mt/gDNA ratio corresponding to different time of blastulation in D5 embryo culture medium (b). According to the patient's age, they were divided into three groups, which are less than or equal to 30 years old, greater than 31 years old and less than 34 years old and greater than or equal to 35 years old (<=30, 31-34 and >=35), such as the abscissa. The ordinate represents the ratio of mt/gDNA in the culture medium. Data are means ± SEM.
ICsI. We used natural cycle ICSIs but did not use FSH for in vitro ovum maturation. We only selected naturally occurring MII (second metaphase) cells for intracytoplasmic sperm injection. A total of 585 eggs were taken from 52 cases, of which 512 oocytes were in MII. The number of eggs taken in each case and the number of oocytes reached MII in each case can be found in Supplement Table 2 Embryo culture was under an oxygen concentration of 5%. In order to allow the embryo culture to reach a temperature suitable for embryonic growth and development, and to bring the pH of the medium to the optimal pH range required by the instructions, we usually incubate the embryo culture media for 10 h before use. On the third day, the embryos that were developed to 6-8 cells were transferred into 25 ul of G-2 PLUS (Ref. No. 10132, Vitrolife, Sweden) containing 5% HSA and cultured until the 5-6th day. The blastocysts were scored according to the method of Gardner et al. 51 . The blastocysts with good scores were selected for transplantation. Figure 9. The mt/gDNA ratio for different pregnancy outcomes. At D3, the mt/gDNA ratio in the embryo culture medium corresponding to different pregnancy outcomes (a). Mt/gDNA ratio corresponding to different pregnancy outcomes in D5 embryo culture medium (b). Based on the pregnancy outcome of the embryo, the samples were divided into two groups, success and failure, such as the abscissa. The 28-day B-ultrasound can monitor the gestational sac and fetal heartbeat as the success criteria. The ordinate of the ordinate represents the ratio of mt/gDNA in the culture medium. Data are means + SEM. ( Negative control (NC). The culture medium identically processed but did not contain embryo was used as a negative control (NC1) and unused culture medium was also used as a negative control (NC2).
WGA and NGs. The MALBAC method was applied to WGA according to manufacture's protocols (catalog no. YK001B; YIKON Genomics). Briefly, amplification begins by annealing DNA to a random primer pool, each with a common 27 nucleotide sequence and 8 variable nucleotides. Prior to amplifying the DNA index to ~2 μg for NGS, a quasi-linear pre-amplification step was performed. Using a Covaris S2 system (Covaris, USA), approximately 400 ng of purified DNA was sonicated into a fragment of approximately 150-250 bp and purified using the Qiagen PCR purification kit (Qiagen, Germany). A sequencing library DNA library preparation was then constructed using NEB Next Ultra (New England Biolabs, UK). The constructed library was sequenced on an Illumina HiSeq2500 platform (Illumina, USA). Data analysis. The MALBAC amplified genome of each sample was sequenced using the Illumina HiSeq. 2500 platform for a total of approximately 1.5 million reads. The NGS data was analyzed and the chromosomal copy number variation was investigated as described in previous reports 48,52,53 . The high quality read numbers were counted along the whole genome with a bin size of 1.5~2 Mb and normalized by the GC content and a reference dataset. An increase of 2 to 3 copies of the copy number results in a 50% increase in the reading of the particular genomic fragment, while a decrease in the copy number from 2 to 1 results in a 50% reduction in the genomic fragment reading. Mitochondrial content is quantified in the form of copy number/nuclear genome. Sequencing reads were mapped to the mitochondrial genome and were counted and normalized to autosomes. Mitochondrial copy numbers per nuclear genome (MCN) were calculated with the following equation (1). Independent sample t test was used to assess the relationship between mt/gDNA and different embryo quality indicators. = * * * MCN autosome mappable region 2 mitochondr ion mapped reads autosome mapped reads mitochondr ion mappable region (1) statistical analyses. The Mann-Whitney test was used to compare the mtDNA/gDNA ratio in spent embryo media and media controls (GraphPad; GraphPad Software). Additionally, it was used to compare differences in mtDNA/gDNA between groups when chromosome euploidy, cystic outcome (means blastulation outcome), and capsular time were different. Independent sample t-test was used to compare differences in mtDNA/ gDNA between groups when ICM score, TE score, fragment ratio, and female age were different (SPSS). The independent sample t-test was also used to compare differences in mtDNA/gDNA between the two groups (n = 12) when pregnancy outcomes were different (SPSS). The results were considered statistically significant at P < 0.05.