P-240 High mitochondrial DNA content in Day6 blastocyst may impair ongoing pregnancy rate
Authors: Barberi M , Ruberti A , Pirastu G , Spinella F , Biricik A , Mencacci C , Greco E 
Published: June 2023

Abstract

Study question
Is there an association between mitochondrial DNA (mtDNA) content and Ongoing Pregnancy Rate (OPR) in D5 or D6 euploid blastocyst?

Summary answer
Day6 OPR is decreased in high mtDNA content blastocyst. Day5 and Day6 mtDNA levels are independent of maternal age or standard morphology.

What is known already
Preimplantation development is an energy-demanding process and mitochondrial ATP production is crucial for cellular activity in fast replicating cells. Mitochondrial content in preimplantation embryo has been proposed as a marker of embryo potential in term of viability and implantation success though the dynamics and distribution of mitochondria in human embryo is still debated. In recent years, it has been suggested that low mtDNA levels were associated with euploid chromosomal complement and higher implantation rate while other studies failed to find a correlation between mtDNA content and reproductive outcome.

Study design, size, duration
This study is a retrospective cohort analysis from 2020 to 2022 including 343 Day5 and 187 D6 single euploid blastocyst transfer. Primary endpoint was OPR beyond pregnancy week 16. Day5 and Day6 frozen-thawed transfer were divided based on blastocyst mtDNA content in four groups: Day5-high, Day5-low, D6-high, D6-low. Secondary endpoints were relationship between mtDNA content, maternal age and standard morphology in all obtained blastocysts. Statistical differences were compared by Chi-Square test.

Participants/materials, setting, methods
771 couples performed IVF cycles with next-generation sequencing (NGS)-preimplantation genetic testing of aneuploidy (PGT-A) (age 19-47yrs; Mean age 37.8yrs). Following culture in sequential media 700 euploid blastocyst were obtained and mitochondrial and chromosomal DNA copy number variation were examined simultaneously with next generation sequencing (NGS) methodology. mtDNA copy numbers was based on the observed ratios of sequence coverages between mtDNA and nuclear DNA.

Main results and the role of chance
In our clinical setting OPR is similar in Day5 vs. Day6 transfers (55% vs. 49% n.s.). However when mtDNA content is considered D6-low OPR is comparable to D5-high or low (57.3%; 53.3%; 57,3% respectively n.s.) while D6-high transfers yield significantly decreased OPR (31,2% p &lt; 0.05). When stratified by age only Advanced Maternal Age (AMA) patients ( &gt; = 38yrs) showed this decrease at a significant level (Day6 high OPR 20% p &lt; 0,05).This result in transferred blastocyst is obtained despite high:low ratio of all biopsied blastocyst is constant in Day5 (1:1) (47%-53%) and markedly decreased in Day6 (1:4) (25%-75%) either overall or in younger (n.s.) and AMA patients (n.s.). Moreover when standard morphology distribution is evaluated according to Gardner classification criteria top, fair and poor quality blastocysts display the same 1:1 high:low blastocysts mtDNA ratio in Day5 and 1:4 in Day 6 overall and in younger and AMA patients. Hence, the mtDNA blastocyst content is independent of patients’ age and blastocyst quality. The origin of Day6 OPR decrease could therefore depend on the number of Day6-high blastocyst transferred in AMA patients and ultimately in the number of blastocyst available to transfer for each patient.

Limitations, reasons for caution
Due to the small numbers of Day6-high mtDNA blastocyst, a larger sample size is required to confirm these preliminary findings. Moreover, the retrospective nature of the study may introduce some bias mainly in patients’ characteristics and the strategy of embryo selected for transfer.

Wider implications of the findings
While Day5-high blastocyst yield higher OPR the persistence of higher mtDNA levels in Day6 blastocyst may hamper their implantation potential. Our findings may help to select the embryo to transfer to maximize transfer success. D6 blastocysts with low mtDNA content should be preferred whenever a choice is possible.

Trial registration number
Not Applicable