Faculty Bibliography

STUDY QUESTION: Does the amount of mitochondrial DNA (mtDNA) in blastocyst biopsy specimens have the potential to serve as a biomarker of euploid embryo implantation ability, independent of morphology? SUMMARY ANSWER: The results of this study strongly suggest that elevated mtDNA levels, above a previously defined threshold, are strongly associated with blastocyst implantation failure and represent an independent biomarker of embryo viability. WHAT IS KNOWN ALREADY: Improved methods of embryo selection are highly desirable in order to increase the efficiency of IVF treatment. At present, even the transfer of chromosomally normal embryos of high morphological grade cannot guarantee that a pregnancy will follow. Recently, it has been proposed that the quantity of mtDNA in embryonic cells may be an indicator of developmental potential, with higher levels of mtDNA associated with reduced implantation. However, thus far reported data sets have been relatively small and in some cases have lacked appropriate validation. STUDY DESIGN, SIZE, DURATION: This large, blinded, retrospective study involved the analysis of relative mtDNA levels in 1505 euploid blastocysts obtained from 490 couples undergoing preimplantation genetic testing for aneuploidy. Implantation outcomes were compared to mtDNA levels in order to determine the capacity of the method to predict viability and to assess the validity of previously established thresholds. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA from blastocyst biopsy samples was amplified and then subjected to aneuploidy analysis using next generation sequencing or array comparative genomic hybridization. Only those embryos classified as chromosomally normal had their mtDNA levels assessed. This analysis was undertaken retrospectively using quantitative real-time PCR, without knowledge of the outcome of embryo transfer. Predictions of implantation failure, based upon mtDNA levels were subsequently compared to the observed clinical results. All cycles involved the transfer of a single embryo. MAIN RESULTS AND THE ROLE OF CHANCE: Of all blastocysts analyzed, 9.2% (139/1505) contained mtDNA levels above a previously established viability threshold and were therefore predicted to have reduced chances of implantation. To the date of analysis, 282 euploid blastocysts had been transferred with an overall implantation rate of 65.6% (185/282). Of the transferred embryos, 249 contained levels of mtDNA in the normal range, 185 of which produced a pregnancy, giving an implantation rate of 74.3% for euploid embryos with 'normal' quantities of mtDNA. However, 33 of the transferred embryos were determined to have elevated mtDNA quantities. None of these led to a pregnancy. Therefore, the negative predictive value of mtDNA assessment in this cohort was 100% (33/33). The difference between the implantation rates for embryos with normal and elevated mtDNA levels was highly significant (P < 0.0001). The mtDNA thresholds, used for classification of embryos, were unaffected by female age or the clinic in which the IVF was undertaken. The probability of an embryo having elevated levels of mtDNA was not influenced by variation in embryo morphology. LIMITATIONS, REASONS FOR CAUTION: This study provides strong evidence that mtDNA quantification can serve as a valuable tool to assist the evaluation of blastocyst viability. However, to determine the true extent of any clinical benefits, other types of investigations, such as non-selection studies and randomized controlled trials, will also be necessary. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study suggest that mtDNA quantity can serve as an independent biomarker for the prediction of euploid blastocyst implantation potential. Prospective studies should now be undertaken to confirm these results. Additionally, investigations into the underlying biological cause(s) of elevated mtDNA levels and an enhanced understanding of how they relate to diminished implantation potential would be invaluable. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by funding provided by Reprogenetics. None of the authors have any competing interests.

PURPOSE: Our objective was to determine if a change in serum P4 from day of transfer (defined as day 19) to day 28 could predict live birth outcome in patients undergoing IVF. METHODS: This study was a retrospective analysis of fresh IVF cycles from 2010 to 2013 at a single center. Primary outcomes include raw and percent change in serum P4, live birth rate, missed abortion, and biochemical pregnancies. RESULTS: Our results showed an association between live birth rate and percent change in P4. Patients with a 10% or greater drop in serum P4 from day 19 to day 28 had a lower live birth rate, at 26 versus 63%. Interestingly, both groups had "normal" serum P4 levels on day 19, but patients with a 10% or greater drop had lower P4 levels than their counterparts. There was no association between percent P4 change and spontaneous abortion or biochemical pregnancy. CONCLUSIONS: This is the first study to show that percent drop in serum P4 from day of transfer to day 28 is associated with decreased rates of live birth and ongoing pregnancy in fresh IVF cycles, even despite "high or normal" P4 levels on day of transfer.

The ovary contains oocytes within immature (primordial) follicles that are fixed in number at birth. Activation of follicles within this fixed pool causes an irreversible decline in reproductive capacity, known as the ovarian reserve, until menopause. Premenopausal women undergoing commonly used genotoxic (DNA-damaging) chemotherapy experience an accelerated loss of the ovarian reserve, leading to subfertility and infertility. Therefore, there is considerable interest but little effective progress in preserving ovarian function during chemotherapy. Here we show that blocking the kinase mammalian/mechanistic target of rapamycin (mTOR) with clinically available small-molecule inhibitors preserves ovarian function and fertility during chemotherapy. Using a clinically relevant mouse model of chemotherapy-induced gonadotoxicity by cyclophosphamide, and inhibition of mTOR complex 1 (mTORC1) with the clinically approved drug everolimus (RAD001) or inhibition of mTORC1/2 with the experimental drug INK128, we show that mTOR inhibition preserves the ovarian reserve, primordial follicle counts, serum anti-Mullerian hormone levels (a rigorous measure of the ovarian reserve), and fertility. Chemotherapy-treated animals had significantly fewer offspring compared with all other treatment groups, whereas cotreatment with mTOR inhibitors preserved normal fertility. Inhibition of mTORC1 or mTORC1/2 within ovaries was achieved during chemotherapy cotreatment, concomitant with preservation of primordial follicle counts. Importantly, our findings indicate that as little as a two- to fourfold reduction in mTOR activity preserves ovarian function and normal birth numbers. As everolimus is approved for tamoxifen-resistant or relapsing estrogen receptor-positive breast cancer, these findings represent a potentially effective and readily accessible pharmacologic approach to fertility preservation during conventional chemotherapy.