Ovarian ageing: the role of mitochondria in oocytes and follicles.

There is a great inter-individual variability of ovarian ageing, and almost 20% of patients consulting for infertility show signs of premature ovarian ageing. This feature, taken together with delayed childbearing in modern society, leads to the emergence of age-related ovarian dysfunction concomitantly with the desire for pregnancy. Assisted reproductive technology is frequently inefficacious in cases of ovarian ageing, thus raising the economic, medical and societal costs of the procedures. Ovarian ageing is characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. Mitochondria play a central role in follicular atresia and could be the main target of the ooplasmic factors determining oocyte quality adversely affected by ageing. Indeed, the oocyte is the richest cell of the body in mitochondria and depends largely on these organelles to acquire competence for fertilization and early embryonic development. Moreover, the oocyte ensures the uniparental transmission and stability of the mitochondrial genome across the generations. This review focuses on the role played by mitochondria in ovarian ageing and on the possible consequences over the generations. PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning mitochondria and ovarian ageing, in animal and human species. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA'; 'ovarian reserve', 'oocyte', 'ovary' or 'cumulus cells'; and 'ageing' or 'ovarian ageing'. These keywords were combined with other search phrases relevant to the topic. References from these articles were used to obtain additional articles. There is a close relationship, in mammalian models and humans, between mitochondria and the decline of oocyte quality with ageing. Qualitatively, ageing-related mitochondrial (mt) DNA instability, which leads to the accumulation of mtDNA mutations in the oocyte, plays a key role in the deterioration of oocyte quality in terms of competence and of the risk of transmitting mitochondrial abnormalities to the offspring. In contrast, some mtDNA haplogroups are protective against the decline of ovarian reserve. Quantitatively, mitochondrial biogenesis is crucial during oogenesis for constituting a mitochondrial pool sufficiently large to allow normal early embryonic development and to avoid the untimely activation of mitochondrial biogenesis. Ovarian ageing also seriously affects the dynamic nature of mitochondrial biogenesis in the surrounding granulosa cells that may provide interesting alternative biomarkers of oocyte quality. A fuller understanding of the involvement of mitochondria in cases of infertility linked to ovarian ageing would contribute to a better management of the disorder in the future.

May-Panloup P ，Boucret L ，Chao de la Barca JM ，Desquiret-Dumas V ，Ferré-L'Hotellier V ，Morinière C ，Descamps P ，Procaccio V ，Reynier P ... -  《-》

Relationship between diminished ovarian reserve and mitochondrial biogenesis in cumulus cells.

What part do mitochondria play in cases of diminished ovarian reserve (DOR)? Mitochondrial biogenesis in cumulus cells may be linked with impaired oocyte competence in patients with DOR. DOR, one of the causes of infertility even in young women, is characterized by the depletion of the ovarian pool associated with a decline in oocyte competence. Mitochondria, which play a role in oocyte quality, could be involved in the pathogenesis of DOR. The study of cumulus cells offers an interesting non-invasive approach for evaluating oocyte quality and the metabolic processes on which it depends. If mitochondrial dysfunction is involved in DOR, it is likely to have an impact on the functioning of cumulus cells. This is an observational study of 74 immature oocyte-cumulus complexes retrieved from 47 women undergoing in vitro fertilization with intracytoplasmic sperm injection at the University Hospital of Angers, France, from March 2013 to March 2014. The women were divided into two groups: one group included 26 women with DOR, and the other, which included 21 women with a normal ovarian reserve (NOR), served as a control group. The oocyte mitochondrial content and the average mitochondrial content of the cumulus cells were assessed by mitochondrial (mt)DNA quantification using a quantitative real-time PCR technique. Microfluidic-based quantitative RT-PCR assays were used to quantify the expression of 13 genes involved in mitochondrial functions such as apoptosis and antioxidant activity or in mitochondrial biogenesis. We used orthogonal partial least-squares discriminant analysis (OPLS-DA) to distinguish between the DOR group and the NOR group of patients, and an OPLS model to predict the value of the oocyte mtDNA content that could be used as a critical marker of oocyte quality. The OPLS-DA model showed a good predictive capability (Q2 = 0.543). Using the variable importance in projection (VIP) metric we found three mitochondrial variables distinguishing the DOR group from the NOR group of patients, i.e. the oocyte mtDNA content (VIP = 0.92), the cumulus cell mtDNA content (VIP = 0.95) and the expression in cumulus cells of peroxisome proliferator-activated receptor γ coactivator 1 alpha (PPARGC-1A) (VIP = 1.10), all of which were lower in the DOR group than in the NOR group of patients. The OPLS model was able to satisfactorily predict the oocyte mtDNA content in only the NOR group of patients (Q2 = 0.506). We found four new variables positively linked to the oocyte mitochondrial mass, i.e. the cumulus cell mtDNA content (VIP = 1.19), and the expression in cumulus cells of three factors of mitochondrial biogenesis: polymerase gamma (POLG) (VIP = 2.13), optic atrophy 1 (OPA1) (VIP = 1.89) and the transcription factor associated with mitochondria (TFAM) (VIP = 1.32). This is a descriptive study. Because of ethical concerns in human clinical practice, this study has been performed only on immature oocytes and corresponding cumulus cells, which are usually discarded during in vitro fertilization procedures. Cumulus cells may govern mitochondrial biogenesis, creating an adequate oocyte mitochondrial pool to promote embryonic development. The alteration of this process in patients with DOR may account for the impairment of oocyte quality. This suggests that some mitochondrial characteristics of cumulus cells may serve as indicators of oocyte competence and that oocyte quality may be improved by products enhancing mitochondrial biogenesis. This work was supported by a grant from the University Hospital of Angers, France: 'Appel d'offre interne à la recherche 2014'. N/A.

Boucret L ，Chao de la Barca JM ，Morinière C ，Desquiret V ，Ferré-L'Hôtellier V ，Descamps P ，Marcaillou C ，Reynier P ，Procaccio V ，May-Panloup P ... -  《-》

Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing.

Does ovarian ageing increase the number of heteroplasmic mitochondrial DNA (mtDNA) point mutations in oocytes? Our results suggest that oocytes are not subject to the accumulation of mtDNA point mutations during ovarian ageing. Ageing is associated with the alteration of mtDNA integrity in various tissues. Primary oocytes, present in the ovary since embryonic life, may accumulate mtDNA mutations during the process of ovarian ageing. This was an observational study of 53 immature oocyte-cumulus complexes retrieved from 35 women undergoing IVF at the University Hospital of Angers, France, from March 2013 to March 2014. The women were classified in two groups, one including 19 women showing signs of ovarian ageing objectified by a diminished ovarian reserve (DOR), and the other, including 16 women with a normal ovarian reserve (NOR), which served as a control group. mtDNA was extracted from isolated oocytes, and from their corresponding cumulus cells (CCs) considered as a somatic cell compartment. The average mtDNA content of each sample was assessed by using a quantitative real-time PCR technique. Deep sequencing was performed using the Ion Torrent Proton for Next-Generation Sequencing. Signal processing and base calling were done by the embedded pre-processing pipeline and the variants were analyzed using an in-house workflow. The distribution of the different variants between DOR and NOR patients, on one hand, and oocyte and CCs, on the other, was analyzed with the generalized mixed linear model to take into account the cluster of cells belonging to a given mother. There were no significant differences between the numbers of mtDNA variants between the DOR and the NOR patients, either in the oocytes (P = 0.867) or in the surrounding CCs (P = 0.154). There were also no differences in terms of variants with potential functional consequences. De-novo mtDNA variants were found in 28% of the oocytes and in 66% of the CCs with the mean number of variants being significantly different (respectively 0.321, SD = 0.547 and 1.075, SD = 1.158) (P < 0.0001). Variants with a potential functional consequence were also overrepresented in CCs compared with oocytes (P = 0.0019). N/A. Limitations may be due to the use of immature oocytes discarded during the assisted reproductive technology procedure, the small size of the sample, and the high-throughput sequencing technology that might not have detected heteroplasmy levels lower than 2%. The alteration of mtDNA integrity in oocytes during ovarian ageing is a recurring question to which our pilot study suggests a reassuring answer. This work was supported by the University Hospital of Angers, the University of Angers, France, and the French national research centers, INSERM and the CNRS. There are nocompeting interests.

Boucret L ，Bris C ，Seegers V ，Goudenège D ，Desquiret-Dumas V ，Domin-Bernhard M ，Ferré-L'Hotellier V ，Bouet PE ，Descamps P ，Reynier P ，Procaccio V ，May-Panloup P ... -  《-》

Maternal ageing impairs mitochondrial DNA kinetics during early embryogenesis in mice.

Does ageing affect the kinetics of the mitochondrial pool during oogenesis and early embryogenesis? While we found no age-related change during oogenesis, the kinetics of mitochondrial DNA content and the expression of the factors involved in mitochondrial biogenesis appeared to be significantly altered during embryogenesis. Oocyte mitochondria are necessary for embryonic development. The morphological and functional alterations of mitochondria, as well as the qualitative and quantitative mtDNA anomalies, observed during ovarian ageing may be responsible for the alteration of oocyte competence and embryonic development. The study, conducted from November 2016 to November 2017, used 40 mice aged 5-8 weeks and 45 mice aged 9-11 months (C57Bl6/CBA F(1)). A total of 488 immature oocytes, with a diameter ranging from 20 μm to more than 80 μm, were collected from ovaries, and 1088 mature oocytes or embryos at different developmental stages (two PN, one-cell, i.e. syngamy, two-cell, four-cell, eight-cell, morula and blastocyst) were obtained after ovarian stimulation and, for embryos, mating. Mitochondrial DNA was quantified by quantitative PCR. We used quantitative reverse transcriptase PCR (RT-PCR) (microfluidic method) to study the relative expression of three genes involved in the key steps of embryogenesis, i.e. embryonic genome activation (HSPA1) and differentiation (CDX2 and NANOG), two mtDNA genes (CYB and ND2) and five genes essential for mitochondrial biogenesis (PPARGC1A, NRF1, POLG, TFAM and PRKAA). The statistical analysis was based on mixed linear regression models applying a logistic link function (STATA v13.1 software), with values of P < 0.05 being considered significant. During oogenesis, there was a significant increase in oocyte mtDNA content (P < 0.0001) without any difference between the two groups of mice (P = 0.73). During the first phase of embryogenesis, i.e. up to the two-cell stage, embryonic mtDNA decreased significantly in the aged mice (P < 0.0001), whereas it was stable for young mice (young/old difference P = 0.015). The second phase of embryogenesis, i.e. between the two-cell and eight-cell stages, was characterized by a decrease in embryonic mtDNA for young mice (P = 0.013) only (young/old difference P = 0.038). During the third phase, i.e. between the eight-cell and blastocyst stage, there was a significant increase in embryonic mtDNA content in young mice (P < 0.0001) but not found in aged mice (young/old difference P = 0.002). We also noted a faster expression of CDX2 and NANOG in the aged mice than in the young mice during the second (P = 0.007 and P = 0.02, respectively) and the third phase (P = 0.01 and P = 0.008, respectively) of embryogenesis. The expression of mitochondrial genes CYB and ND2 followed similar kinetics and was equivalent for both groups of mice, with a significant increase during the third phase (P < 0.01). Of the five genes involved in mitochondrial biogenesis, i.e. PPARGC1A, NRF1, POLG, TFAM and PRKAA, the expression of three genes decreased significantly during the first phase only in young mice (NRF1, P = 0.018; POLGA, P = 0.002; PRKAA, P = 0.010), with no subsequent difference compared to old mice. In conclusion, during early embryogenesis in the old mice, we suspect that the lack of a replicatory burst before the two-cell stage, associated with the early arrival at the minimum threshold value of mtDNA, together with the absence of an increase of mtDNA during the last phase, might potentially deregulate the key stages of early embryogenesis. N/A. Because of the ethical impossibility of working on a human, this study was conducted only on a murine model. As superovulation was used, we cannot totally exclude that the differences observed were, at least partially, influenced by differences in ovarian response between young and old mice. Our findings suggest a pathophysiological explanation for the link observed between mitochondria and the deterioration of oocyte quality and early embryonic development with age. This work was supported by the University of Angers, France, by the French national research centres INSERM and the CNRS and, in part, by PHASE Division, INRA. There are no competing interests.

May-Panloup P ，Brochard V ，Hamel JF ，Desquiret-Dumas V ，Chupin S ，Reynier P ，Duranthon V ... -  《-》

Mitochondrial SIRT3 and its target glutamate dehydrogenase are altered in follicular cells of women with reduced ovarian reserve or advanced maternal age.

Pacella-Ince L ，Zander-Fox DL ，Lan M 《-》