Does supplementation with mitochondria improve oocyte competence? A systematic review

Mitochondrial supplementation was proposed as a complementary treatment to assisted reproductive technologies to improve oocyte competence and support post-fertilization development. This strategy is based on the fact that poor-quality/aged oocytes contain lower and dysfunctional mitochondria. However, the efficacy and safety of mitochondrial supplementation is still controversial. Therefore, this review summarizes the clinical/biological outcomes of mitochondrial supplementation, aiming to improve oocyte competence or explore the safety of this technique, and was based on an online search using PubMed and Web of Science, until September 2019. The studies included reported outcomes related to efficacy and safety of mitochondrial supplementation either in human or animal models (bovine, porcine and mouse). Extracted data were organized according to study objective, the mitochondrial source and the main outcomes: fertilization/pregnancy rates, embryo development and adverse outcomes. Clinical pregnancy was not improved in the only randomized controlled trial published, although an increase was demonstrated in other non-randomized studies. Fertilization rate and embryo development were not different from control groups in the majority of studies, although performed in different contexts and using diverse sources of mitochondria. The safety of mitochondria transfer is still a concern, however, the euploid rate and the absence of reported congenital malformation from the clinical studies are reassuring. In summary, mitochondrial supplementation does not seem to cause harm although the benefit of improving oocyte competence is still unclear due to the diversity of methodological approaches and low-quality of the data available. Analyzed data supports the need to investigate further, in both pre-clinical and clinical contexts.

The obligate need for accuracy in reporting preclinical studies relevant to clinical trials: autologous germline mitochondrial supplementation for assisted human reproduction as a case study

A now large body of work has solidified the central role that mitochondria play in oocyte development, fertilization, and embryogenesis. From these studies, a new technology termed autologous germline mitochondrial energy transfer was developed for improving pregnancy success rates in assisted reproduction. Unlike prior clinical studies that relied on the use of donor, or nonautologous, mitochondria for microinjection into eggs of women with a history of repeated in vitro fertilization failure to enhance pregnancy success, autologous germline mitochondrial energy transfer uses autologous mitochondria collected from oogonial stem cells of the same woman undergoing the fertility treatment. Initial trials of autologous germline mitochondrial energy transfer during - in vitro fertilization at three different sites with a total of 104 patients indicated a benefit of the procedure for improving pregnancy success rates, with the birth of children conceived through the inclusion of autologous germline mitochondrial energy transfer during in vitro fertilization. However, a fourth clinical study, consisting of 57 patients, failed to show a benefit of autologous germline mitochondrial energy transfer– in vitro fertilization versus in vitro fertilization alone for improving cumulative live birth rates. Complicating this area of work further, a recent mouse study, which claimed to test the long-term safety of autologous mitochondrial supplementation during in vitro fertilization, raised concerns over the use of the procedure for reproduction. However, autologous mitochondria were not actually used for preclinical testing in this mouse study. The unwarranted fears that this new study’s erroneous conclusions could cause in women who have become pregnant through the use of autologous germline mitochondrial energy transfer during- in vitro fertilization highlight the critical need for accurate reporting of preclinical work that has immediate bearing on human clinical studies.

163 DEVELOPMENT OUTCOME AND MITOCHONDRIAL MEMBRANE POTENTIAL IN BOVINE PARTHENOTES SUBMITTED TO MITOCHONDRIAL SUPPLEMENTATION AND DEPLETION AT THE 1-CELL STAGE

Apart from supplying ATP for cellular function by establishing a proton gradient across the inner membrane (Ψmm), mitochondria also play a central role in death pathways of most cells in the organism. Since mitochondria undergo major transformations during early embryogenesis, we hypothesize that the amount of these organelles at the 1-cell stage is correlated to development. Herein we evaluated the impact of mitochondrial supplementation and depletion on embryonic development and Ψmm. Immature oocytes were collected from ovaries of slaughtered cows and submitted to in vitro maturation for 26 h and parthenogenetically activated by 5 min exposure to 5 μM ionomycin followed by 3 h culture in 2 mM 6-DMAP (control, C). In order to deplete mitochondria (depletion, D), zygotes were centrifuged (15 min 10,000g in HEPES-SOF with cytochalasin B) and the mitochondria, at the mitochondria-enriched cytoplast fraction (MECF), were partially removed by micro-manipulation. Mitochondria supplementation was performed by introducing the MECF from the depleted zygote into another zygote (supplemented, S). Centrifugation and cytoplasm removal effects (7.1% of volume) were tested using 2 other control groups: centrifuged zygotes (centrifuged control, CC) and aspirated zygotes (aspirated control, AC). Embryos were submitted to Mitotracker CMXRos (500 ηM) staining to measure Ψmm levels. Zygotes were cultured in SOF medium for 3, 72, and 168 h in 5% O2, 5% CO2, and 90% N2. Blastocysts were fixed and stained with Hoechst 33342 to count nuclei. At 72 h, development to the 8-cell stage was reduced in CC (41.6%), D (37.0%), and S (33.2%) groups when compared to C (52.5%) and AC (49.3%) control groups (P < 0.05, chi-square). Similar findings were observed at 168 h where the CC (31.9%), D (21.0%), and S (19.1%) group showed reduced development to the blastocyst stage when compared to the C (40.5%) and AC (41.8) control groups (P > 0.05, chi-square). Moreover, nuclear number was significantly reduced in the D (101.6 ± 12.4) and S (102.2 ± 7.8) groups when compared to the C (137.9 ± 6.7) and CC (126.6 ± 7.4) controls. The AC group (112.2 ± 7.8) is similar to CC, D, and S. These results indicate a sensitivity of embryos to changes (i.e. supplementation and depletion) in the number of mitochondria present at the 1-cell stage on further development to the 8-cell and blastocyst stages. With the exception of Ψmm of AC blastocysts at 168 (62.8 ± 6.8, P < 0.05; the Tuckey test), all other controls and treated blastocysts showed similar Ψmm levels (100.0 ± 6.2; 84.1 ± 8.3; 85.9 ± 6.6; 97.1 ± 19.1, respectively for C, CC, D, and S groups). Although, these results indicate that the Ψmm is not affected by the removal or addition of mitochondria. A reduction in cytoplasm volume (only the AC group) seems to adversely affect the ability of mitochondria to sustain normal Φmm levels at the blastocyst stage without affecting development or nuclear number. Together, supplementation and depletion of MECF are detrimental to development through mechanisms independent of mitochondrial Ψmm. Financial support: FAPESP-Brazil.