Mitochondrial DNA content reduction in the most fertile spermatozoa is accompanied by increased mitochondrial DNA rearrangement.

STUDY QUESTION: Is there an association between male fertility and spermatozoa mitochondrial DNA (mtDNA) copy number and genome rearrangements? SUMMARY ANSWER: Normal spermatozoa not only have a lower mtDNA copy number but also more DNA rearrangements than spermatozoa of men with severe oligoasthenospermia (SOA). WHAT IS KNOWN ALREADY: While there is a consensus that mtDNA content is decreased in the most fertile spermatozoa, the role of mtDNA sequence alteration in male infertility is unclear. High-throughput sequencing, which allows an exhaustive analysis of mtDNA rearrangements and mutations, could be helpful in this context, but has yet to be used. STUDY DESIGN, SIZE, DURATION: This is an observational study of semen samples obtained from 44 men undergoing ART at an academic infertility centre in France, from October 2018 to November 2020. The men were classified into two groups: 20 men in the SOA group and 24 men with normal semen parameters in the control group. PARTICIPANTS/MATERIALS, SETTING, METHODS: For each patient and control, mtDNA was isolated from sperm fractions from the 40% and 90% layers of the density gradient. The average mtDNA content of each sample was assessed using digital PCR. Deep sequencing was performed using next-generation sequencing. Signal processing and base calling were performed via the embedded pre-processing pipeline, the variants were analysed using an in-house workflow and a dedicated tool, based on soft-clipping, was used to study large mtDNA rearrangements. The distribution and the type of rearrangements and variants were compared between patients with SOA and controls on one hand, and between the 40% and 90% gradient layers, on the other hand. MAIN RESULTS AND THE ROLE OF CHANCE: The mtDNA content of spermatozoa in the SOA group was significantly higher than in the control group (P < 0.0001). Moreover, mtDNA content was significantly higher in spermatozoa from the 40% layer (the most fertile spermatozoa) compared to the 90% layer, both in the SOA (P = 0.02) and the control group (P < 0.0001). The frequency of large mtDNA deletions and duplications was significantly higher in the control group (P = 0.002). Most of these rearrangements are potentially related to DNA breaks and their number was reduced by the removal of the linear mtDNA from the samples. Heteroplasmic variants were found more frequently in the SOA group (P = 0.05) and in the 40% layer (P = 0.03), but none had any obvious functional consequence. LIMITATIONS, REASONS FOR CAUTION: Our findings are novel and significant but should be verified in larger cohorts and other types of male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that sperm mtDNA rearrangements are not necessarily associated with mitochondrial dysfunction and male infertility. Instead, they seem to be concomitant with the process of mtDNA content reduction in the most potentially fertile spermatozoa. Furthermore, they refute the hypothesis that, in the case of mtDNA alteration, a compensatory mechanism allows an increase in mtDNA copy number to rectify the energy deficit. The increased frequency of mtDNA rearrangements in the most fertile spermatozoa is a novel result that offers new insight into the relation between sperm quality and mtDNA. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Angers University Hospital (grant AOI CHU Angers 2018), Angers University and the French national research centres INSERM and CNRS. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

The mitochondrial DNA content of cumulus cells may help predict embryo implantation.

PURPOSE: The quantification of mtDNA in cumulus granulosa cells (CGCs) surrounding an oocyte has been positively linked with morphological embryonic quality. In the present study, we evaluated the link between the amount of mtDNA in CGCs surrounding an oocyte and the chances for the corresponding embryo of implanting and leading to an ongoing pregnancy. METHODS: This is an observational study, performed on 84 oocyte-cumulus-complexes (OCCs) having led to the replacement of an embryo in the maternal uterus, retrieved from 71 patients undergoing IVF with intracytoplasmic sperm. The OCCs were classified in two groups, one including 26 OCCs having led to an implanted embryo and the other including 58 OCCs having led to a non-implanted embryo. The average mtDNA content of CGCs was assessed by using a quantitative real-time PCR technique. RESULTS: Significantly higher mtDNA copy numbers in CGCs were associated with implanted embryos than with non-implanted embryos (mean 215 [sd 375] and 59 [sd 72], respectively; p < 104). Multivariate analysis, taking into account the women's age, the embryo quality, and the AMH level, suggests an independent relationship between the mtDNA content of CGCs and the potential of embryo implantation. CONCLUSION: During in vitro fertilization (IVF) procedures, the probability of the implantation of the embryo appears to be closely correlated to the mtDNA copy numbers in the CGCs. Our results highlight the interest of mtDNA quantification in GCGs as a biomarker of the potential of embryo implantation.

The mitochondrial DNA content of cumulus granulosa cells is linked to embryo quality.

STUDY QUESTION: Could the mitochondrial DNA (mtDNA) content of cumulus granulosa cells (CGCs) be related to oocyte competence? SUMMARY ANSWER: The quality of embryos obtained during IVF procedures appears to be linked to mtDNA copy numbers in the CGCs. WHAT IS KNOWN ALREADY: Oocyte quality is linked to oocyte mtDNA content in the human and other species, and the mtDNA copy number of the oocyte is related to that of the corresponding CGCs. Moreover, the quantification of CGC mtDNA has recently been proposed as a biomarker of embryo viability. STUDY DESIGN SIZE, DURATION: An observational study was performed on 452 oocyte-cumulus complexes retrieved from 62 patients undergoing ICSI at the ART Center of the University Hospital of Angers, France, from January to May 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: The average mtDNA content of CGCs was assessed by using a quantitative real-time PCR technique. The relationship between CGC mtDNA content and oocyte maturity and fertilizability, on one hand, and embryo quality, on the other, was investigated using univariate and multivariate generalized models with fixed and mixed effects. MAIN RESULTS AND THE ROLE OF CHANCE: No relationship was found between CGC mtDNA content and oocyte maturity or fertilizability. In contrast, there was a significant link between the content of mtDNA in CGCs surrounding an oocyte and the embryo quality, with significantly higher mtDNA copy numbers being associated with good quality embryos compared with fair or poor quality embryos [interquartile range, respectively, 738 (250-1228) and 342 (159-818); P = 0.006]. However, the indication provided by the quantification of CGC mtDNA concerning the eventuality of good embryo quality was seriously subject to patient effect (AUC = 0.806, 95%CI = 0.719-0.869). The quantity of CGC mtDNA was influenced by BMI and smoking. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The quantification of CGC mtDNA may indicate embryo quality. However, since it is affected by patient specificity, it should be used with caution. It remains to be seen whether this marker could directly predict the implantation capacity of the embryo, which is the main objective in IVF practice. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests that the quantification of CGC mtDNA may be a novel biomarker of embryo viability. However, patient specificity makes it impossible to establish a general threshold value, valid for all patients. Nevertheless, further studies are needed to determine whether the quantification of CGC mtDNA may, in combination with the morpho-kinetic method, offer an additional criterion for selecting the best embryo for transfer from a given cohort. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the University Hospital of Angers, the University of Angers, France, and the French national research centres INSERM and the CNRS. There were no competing interests.

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

STUDY QUESTION: Does ovarian ageing increase the number of heteroplasmic mitochondrial DNA (mtDNA) point mutations in oocytes? SUMMARY ANSWER: Our results suggest that oocytes are not subject to the accumulation of mtDNA point mutations during ovarian ageing. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: 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%. WIDER IMPLICATIONS OF THE FINDINGS: The alteration of mtDNA integrity in oocytes during ovarian ageing is a recurring question to which our pilot study suggests a reassuring answer. STUDY FUNDING/COMPETING INTEREST(S): 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.

Targeted metabolomics reveals reduced levels of polyunsaturated choline plasmalogens and a smaller dimethylarginine/arginine ratio in the follicular fluid of patients with a diminished ovarian reserve.

STUDY QUESTION: Does the metabolomic profile of the follicular fluid (FF) of patients with a diminished ovarian reserve (DOR) differ from that of patients with a normal ovarian reserve (NOR)? SUMMARY ANSWER: The metabolomic signature of the FF reveals a significant decrease in polyunsaturated choline plasmalogens and methyl arginine transferase activity in DOR patients compared to NOR patients. WHAT IS KNOWN ALREADY: The composition of the FF reflects the exchanges between the oocyte and its microenvironment during its acquisition of gametic competence. Studies of the FF have allowed identification of biomarkers and metabolic pathways involved in various pathologies affecting oocyte quality, but no large metabolomic analysis in the context of ovarian ageing and DOR has been undertaken so far. STUDY DESIGN, SIZE, DURATION: This was an observational study of the FF retrieved from 57 women undergoing in vitro fertilization at the University Hospital of Angers, France, from November 2015 to September 2016. The women were classified in two groups: one including 28 DOR patients, and the other including 29 NOR patients, serving as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were enrolled in the morning of oocyte retrieval after ovarian stimulation. Once the oocytes were isolated for fertilization and culture, the FF was pooled and centrifuged for analysis. A targeted quantitative metabolomic analysis was performed using high-performance liquid chromatography coupled with tandem mass spectrometry, and the Biocrates Absolute IDQ p180 kit. The FF levels of 188 metabolites and several sums and ratios of metabolic significance were assessed by multivariate and univariate analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 136 metabolites were accurately quantified and used for calculating 23 sums and ratios. Samples were randomly divided into training and validation sets. The training set, allowed the construction of multivariate statistical models with a projection-supervised method, i.e. orthogonal partial least squares discriminant analysis (OPLS-DA), applied to the full set of metabolites, or the penalized least absolute shrinkage and selection operator with logistic regression (LASSO-LR), applied to the ratios and sums of the metabolites. Both multivariate models showed good predictive performances when applied to the validation set. The final penalized model retained the three most significant variables, i.e. the total dimethylarginine-to-arginine ratio (Total DMA/Arginine), the sum of the polyunsaturated choline plasmalogens (PUFA ae), and the patient's age. The negative coefficients of Total DMA/Arginine and PUFA ae indicated that these FF variables had lower values in DOR patients than in NOR patients. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This study presents two limitations. First, with this targeted metabolomics analysis, we have explored only a limited portion of the FF metabolome. Second, although the signature found was highly significant, the mechanism underlying the dysfunction remains undetermined. WIDER IMPLICATIONS OF THE FINDINGS: The understanding of the mechanisms implied in ovarian ageing is essential for providing an adequate response to affected women desiring pregnancy. Our study proposes an incoming signature that may open new paths towards this goal. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the University Hospital of Angers, the University of Angers, and the French national research centers, INSERM and the CNRS. There were no competing interests.

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

STUDY QUESTION: What part do mitochondria play in cases of diminished ovarian reserve (DOR)? SUMMARY ANSWER: Mitochondrial biogenesis in cumulus cells may be linked with impaired oocyte competence in patients with DOR. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LIMITATIONS, REASONS OF CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant from the University Hospital of Angers, France: 'Appel d'offre interne à la recherche 2014'. TRIAL REGISTRATION NUMBER: N/A.

Surgical treatment of septate uterus in cases of primary infertility and before assisted reproductive technologies.

Septate uterus is the most common congenital uterine malformation in women with infertility. Several criteria are available for the definition of septate uteri, such as the one proposed by the European Society of Human Reproduction and Embryology (ESHRE)/European Society for Gynecological Endoscopy (ESGE) (ESHRE/ESGE), or by the American Society for Reproductive Medicine (ASRM), with notable differences between the two. Recently, a simplified classification was proposed by the Congenital Uterine Malformations Experts (CUME), where a septum is defined as an internal indentation depth≥10mm. To date, there is no consensus on the management of women with a septate uterus and infertility. We have performed an extensive literature appraisal and reviewed all the available international guidelines in order to propose a management strategy for infertile patients with a uterine septum. Hysteroscopic septum incision seems to improve natural conception rates in the year following surgery. Moreover, it improves in vitro fertilization (IVF) outcomes when performed before the embryo transfer, by improving embryo implantation rates. On the other hand, for patients with an arcuate uterus (indentation<1.5cm according to the ASRM guidelines) and infertility, it seems that assisted reproductive technologies are the most appropriate first line treatment. However, in cases of recurrent implantation failure or recurrent pregnancy loss following IVF, hysteroscopic section could be proposed. Overall, we recommend hysteroscopic septum incision for patients with primary infertility, and for patients undergoing assisted reproductive technologies.

[In vitro fertilization versus conversion to intrauterine insemination in patients with poor response to controlled ovarian hyperstimulation]. / Réponse insuffisante à la stimulation en vue de FIV : maintenir la ponction ou choisir l' insémination ?

In women undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF), a poor ovarian response, defined as three of fewer mature follicles, can lead to cancellation of the cycle. However, in women with at least one patent tube and normal semen parameters, conversion to intrauterine insemination (IUI) is considered an option, offering reasonable pregnancy rates at a lower cost and without the complications associated with oocyte retrieval. Studies have shown that in cycles with only one mature follicle, IVF should be canceled. However, in cycles with 2 or 3 mature follicles, patients have the choice between IVF and conversion to IUI. Some studies have shown that IVF is superior to IUI in such cases, whereas other reports failed to find any difference. Most of these studies are retrospective and limited by the presence of several biases and low numbers of cycles, and to this date, there is no consensus on the best approach. We have thus designed a multicenter, randomized non-inferiority study, comparing live birth rates following conversion to IUI or IVF in patients with 2 or 3 mature follicles in COH cycles. Nine hundred and forty patients will be randomized on trigger day to either IVF or conversion to IUI. Our study will also include a medico-economic analysis.

Mitochondrial macro-haplogroup JT may play a protective role in ovarian ageing.

This study of 200 Caucasian women shows that the distribution of the mtDNA macro-haplogroups in patients with diminished ovarian reserve (DOR) differed significantly from that of patients with normal ovarian reserve (NOR) (p=0.02). The JT macro-haplogroup was significantly under-represented in DOR patients compared with NOR patients (p=0.006) and compared with the estimated frequency of 18.8% in the general French population (p=0.0012). Our findings suggest that the risk of a prematurely depleted ovarian reserve would be three times lower for patients carrying the JT macro-haplogroup than for patients with any of the other mtDNA haplogroups (odds ratio: 0.3; 95% CI: 0.13-0.74). If these preliminary results are confirmed in larger independent studies, they should lead to the better management of infertility.