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Reproductive senescence impairs the energy metabolism of human luteinized granulosa cells

Open AccessPublished:August 11, 2021DOI:https://doi.org/10.1016/j.rbmo.2021.08.006

      Abstract

      Research question

      Female age is the single greatest factor influencing reproductive performance and granulosa cells are considered as potential biomarkers of oocyte quality. Is there an age effect on the energy metabolism of human mural granulosa cells?

      Design

      Observational prospective cohort and experimental study including 127 women who had undergone IVF cycles. Women were allocated to two groups: a group of infertile patients aged over 38 years and a control group comprising oocyte donors aged less than 35 years. Individuals with pathologies that could impair fertility were excluded from both groups. Following oocyte retrieval, cumulus and granulosa cells were isolated and their bioenergetic properties (oxidative phosphorylation parameters, rate of aerobic glycolysis and adenine nucleotide concentrations) were analysed and compared.

      Results

      Human mural luteinized granulosa and cumulus cells present high rates of aerobic glycolysis that cannot be increased further when mitochondrial ATP synthesis is inhibited. Addition of follicular fluid to the experimental media is necessary to reach the full respiratory capacity of the cells. Granulosa cells from aged women present lower mitochondrial respiration (12.8 ± 1.6 versus 11.2 ± 1.6 pmol O2/min/mg; P = 0.046), although mitochondrial mass is not decreased, and lower aerobic glycolysis, than those from young donors (12.9 ± 1.3 versus 10.9 ± 0.5 mpH/min/mg; P = 0.009). The concurrent decrease in the two energy supply pathways leads to a decrease in the cellular energy charge (0.87 ± 0.01 versus 0.83 ± 0.02; P < 0.001).

      Conclusions

      Human mural luteinized granulosa cells exhibit a reduction in their energy metabolism as women age that is likely to influence female reproductive potential.

      KEYWORDS

      Introduction

      Female age is the single greatest factor influencing the reproductive performance of couples undergoing infertility treatments (
      • Igarashi H.
      • Takahashi T.
      • Nagase S.
      Oocyte aging underlies female reproductive aging: biological mechanisms and therapeutic strategies.
      ). The 2016 final report from the Society for Assisted Reproductive Technology (SART, https://www.cdc.gov/art/pdf/2016-report/ART-2016-National-Summary-Report.pdf) shows that nearly half of patients under 35 years undergoing an IVF cycle will achieve a live birth using their own eggs, whereas less than 26% of women aged over 37 years will. This becomes even more dramatic beyond 42 years old, with cumulative live birth rates as low as 3.7% (

      Centers for Disease Control and Prevention, American Society for Reproductive Medicine, Society for Assisted Reproductive Technology. 2015 Assisted Reproductive Technology National Summary Report. Atlanta: US Dept of Health and Human Services; 2017.https://www.cdc.gov/art/pdf/2015-report/ART-2015-National-Summary-Report.pdf

      ). Poor oocyte competence is the primary cause of age-related deterioration of reproductive capacity (
      • Navot D.
      • Bergh R.A.
      • Williams M.A.
      • Garrisim G.J.
      • Guzman I.
      • Sandler B.
      • Grunfeld L.
      Poor oocyte quality rather than implantation failure as a cause of age-related decline in female fertility.
      ). Indeed, oocyte donation cycles sustain a live birth rate of 50% irrespective of the woman's age (

      Centers for Disease Control and Prevention, American Society for Reproductive Medicine, Society for Assisted Reproductive Technology. 2015 Assisted Reproductive Technology National Summary Report. Atlanta: US Dept of Health and Human Services; 2017.https://www.cdc.gov/art/pdf/2015-report/ART-2015-National-Summary-Report.pdf

      ).
      Research on mitochondrial biology and cellular bioenergetics has gained increasing attention in recent years, providing new opportunities in different fields of medicine (
      • Picard M.
      • Wallace D.C.
      • Burelle Y.
      The rise of mitochondria in medicine.
      ). Thus, the role of mitochondria in cellular senescence and human ageing has raised considerable interest (
      • Bratic A.
      • Larsson N.G.
      The role of mitochondria in aging.
      ;
      • Sun N.
      • Youle R.J.
      • Finkel T.
      The mitochondrial basis of aging.
      ;
      • Ziegler D.V.
      • Wiley C.D.
      • Velarde M.C.
      Mitochondrial effectors of cellular senescence: beyond the free radical theory of aging.
      ). It is known that mitochondrial dysfunction underlies some of the fertility defects in humans and that mitochondria play a critical role in oocyte quality and early embryo development (
      • Cecchino G.N.
      • Seli E.
      • Alves da Motta E.L.
      • García-Velasco J.A.
      The role of mitochondrial activity in female fertility and assisted reproductive technologies: overview and current insights.
      ;
      • Demain L.A.M.
      • Conway G.S.
      • Newman W.G.
      Genetics of mitochondrial dysfunction and infertility.
      ;
      • Kasapoglu I.
      • Seli E.
      Mitochondrial dysfunction and ovarian aging.
      ;
      • May-Panloup P.
      • Chretien M.F.
      • Malthiery Y.
      • Reynier P.
      Mitochondrial DNA in the oocyte and the developing embryo.
      ). In fact, mitochondria have been suggested as a promising biomarker for IVF outcomes (
      • Kim J.
      • Seli E.
      Mitochondria as a biomarker for IVF outcome.
      ;
      • Legro R.S.
      Introduction: stimulate the mitochondria! A Bonington approach to improving assisted reproductive technology outcomes or a call to action?.
      ). Furthermore, the possibility of overcoming mitochondrial dysfunction to improve oocyte quality and age-related infertility has become the goal of numerous studies (
      • Ben-Meir A.
      • Burstein E.
      • Borrego-Alvarez A.
      • Chong J.
      • Wong E.
      • Yavorska T.
      • Naranian T.
      • Chi M.
      • Wang Y.
      • Bentov Y.
      • Alexis J.
      • Meriano J.
      • Sung H.-K.
      • Gasser D.L.
      • Moley K.H.
      • Hekimi S.
      • Casper R.F.
      • Jurisicova A.
      Coenzyme Q10 restores oocyte mitochondrial function and fertility during reproductive aging.
      ,
      • Ben-Meir A.
      • Kim K.
      • McQuaid R.
      • Esfandiari N.
      • Bentov Y.
      • Casper R.F.
      • Jurisicova A.
      Co-enzyme q10 supplementation rescues cumulus cells dysfunction in a maternal aging model.
      ;
      • Bentov Y.
      • Esfandiari N.
      • Burstein E.
      • Casper R.F.
      The use of mitochondrial nutrients to improve the outcome of infertility treatment in older patients.
      ;
      • Cagnone G.L.M.
      • Tsai T.S.
      • Makanji Y.
      • Matthews P.
      • Gould J.
      • Bonkowski M.S.
      • Elgass K.D.
      • Wong A.S.A.
      • Wu L.E.
      • McKenzie M.
      • Sinclair D.A.
      • St John J.C.
      Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency.
      ;
      • Labarta E.
      • de los Santos M.J.
      • Escribá M.J.
      • Pellicer A.
      • Herraiz S.
      Mitochondria as a tool for oocyte rejuvenation.
      ).
      Ovarian bioenergetics includes a sophisticated metabolic synergism between oocytes and granulosa cells, which is crucial for oocyte maturation during follicular growth (
      • Canipari R.
      Oocyte–granulosa cell interactions.
      ;
      • Cinco R.
      • Digman M.A.
      • Gratton E.
      • Luderer U.
      Spatial characterization of bioenergetics and metabolism of primordial to preovulatory follicles in whole ex vivo murine ovary.
      ). However, little is known about the energy metabolism of human granulosa cells and its impact on IVF outcomes. In the field of assisted reproductive technology (ART), previous studies that intended to explain defects in meiosis and other cellular processes affecting oocyte maturation, fertilization or embryo development have often focused either on the analysis of mitochondrial DNA (mtDNA) as an indicator of mitochondrial content/function (
      • Cecchino G.N.
      • Garcia-Velasco J.A.
      Mitochondrial DNA copy number as a predictor of embryo viability.
      ;
      • Cecchino G.N.
      • Seli E.
      • Alves da Motta E.L.
      • García-Velasco J.A.
      The role of mitochondrial activity in female fertility and assisted reproductive technologies: overview and current insights.
      ;
      • May-Panloup P.
      • Chretien M.F.
      • Malthiery Y.
      • Reynier P.
      Mitochondrial DNA in the oocyte and the developing embryo.
      ;
      • Reynier P.
      • May-Panloup P.
      • Chrétien M.F.
      • Morgan C.J.
      • Jean M.
      • Savagner F.
      • Barrière P.
      • Malthièry Y.
      Mitochondrial DNA content affects the fertilizability of human oocytes.
      ) or on ATP concentrations as an indicator of the cellular energy status (
      • Dalton C.M.
      • Szabadkai G.
      • Carroll J.
      Measurement of ATP in single oocytes: impact of maturation and cumulus cells on levels and consumption.
      ;
      • Downs S.M.
      The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocyte.
      ;
      • Hsu A.L.
      • Townsend P.M.
      • Oehninger S.
      • Castora F.J.
      Endometriosis may be associated with mitochondrial dysfunction in cumulus cells from subjects undergoing in vitro fertilization-intracytoplasmic sperm injection, as reflected by decreased adenosine triphosphate production.
      ;
      • Pasquariello R.
      • Ermisch A.F.
      • Silva E.
      • McCormick S.
      • Logsdon D.
      • Barfield J.P.
      • Schoolcraft W.B.
      • Krisher R.L.
      Alterations in oocyte mitochondrial number and function are related to spindle defects and occur with maternal aging in mice and humans.
      ;
      • Zeng H.T.
      • Ren Z.
      • Yeung W.S.B.
      • Shu Y.M.
      • Xu Y.W.
      • Zhuang G.L.
      • Liang X.Y.
      Low mitochondrial DNA and ATP contents contribute to the absence of birefringent spindle imaged with PolScope in in vitro matured human oocytes.
      ;
      • Zhao J.
      • Li Y.
      Adenosine triphosphate content in human unfertilized oocytes, undivided zygotes and embryos unsuitable for transfer or cryopreservation.
      ). Bioenergetics provides tools to analyse the factors that may lead to alterations in cellular energy levels. Therefore, this study aimed to characterize the bioenergetic profile of human luteinized granulosa cells in order to detect the potential impact of ageing on energy metabolism. It demonstrates that aged cells show a significant decrease in the two main ATP supply pathways, leading to deficient cellular energy charge.

      Materials and methods

       Study design and population

      Observational prospective cohort including 127 women who had undergone IVF and intracytoplasmic sperm injection after ovarian stimulation (Table 1). Patients were allocated to two age groups: the control group consisted of 84 oocyte donors aged under 35, whereas the advanced reproductive age (ARA) group included 43 infertile women aged over 38 years. For the two groups, the following exclusion criteria were adopted: relevant systemic diseases, alcohol or drug abuse, heritable or chromosomal disorders, known mitochondrial dysfunction, anatomical defects of the reproductive system, formal contraindication to pregnancy or ovarian stimulation, prior exposure to radiation or chemotherapy and a body mass index (BMI) ≥30 kg/m2. Among the diseases that could potentially impair fertility and mitochondrial function, the following were excluded in both groups: endocrine abnormalities, recurrent miscarriages, neurodegenerative and heart diseases, polycystic ovary syndrome, endometriosis, infectious and sexually transmitted disorders, and neoplasms. Thus, the group of women aged over 38 basically comprised infertile women due to male factor or unexplained infertility in which the presumed cause was the advanced age.
      Table 1Baseline characteristics, cycle parameters and ovarian response to ovarian stimulation
      CharacteristicControl group (n = 43)ARA group (n = 84)P-value
      Age (years)24.1 ± 3.940.8 ± 2.0<0.001
      BMI (kg/m2)22.4 ± 3.022.9 ± 2.70.323
      AMH (ng/ml)Not determined
      The AMH concentrations of egg donors are not routinely quantified at the study institution.
      1.27 ± 0.95
      AFC19.0 ± 4.49.0 ± 4.2<0.001
      Oestradiol concentration (pg/ml)2452 (288–9847)1729 (285–5844)0.080
      Days of stimulation11 (8–17)10 (7–16)0.711
      Gonadotrophin dose (IU)2000 (775–4800)2250 (1350–4275)<0.001
      Number of oocytes19 (9–54)8 (2–25)<0.001
      Number of mature oocytes14 (5–41)6 (1–23)<0.001
      Oocyte maturity rate0.78 ± 0.130.80 ± 0.180.260
      AFC = antral follicle count; AMH = anti-Müllerian hormone; ARA = advanced reproductive age; BMI = body mass index.
      a The AMH concentrations of egg donors are not routinely quantified at the study institution.

       Serum hormonal measurements

      Serum anti-Müllerian hormone (AMH) quantification was performed prior to ovarian stimulation. Blood samples were obtained by venipuncture. The blood samples collected were allowed to clot for 20 min and were then centrifuged for 10 min at 6000g. Serum samples were analysed by chemiluminescence using a cobas e411 analyser (Roche Diagnostics, Sussex, UK). The analytical sensitivity of the AMH assay was 0.01 ng/ml and the coefficient of variation was below 5%. The analytical sensitivity of the oestradiol assay was <20 pg/ml and the coefficient of variation was below 6%, and for progesterone it was <0.05 ng/ml and below 7%, respectively.

       Ovarian stimulation protocol and ovum retrieval

      In all cases an antagonist protocol was used to carry out ovarian stimulation. Individualized doses of gonadotrophins were determined by an experienced specialist physician. Transvaginal ultrasound for cycle monitoring was performed every 2 days starting on stimulation day 5. Fixed daily doses of 0.25 mg of gonadotrophin-releasing hormone (GnRH) antagonist (Orgalutran, Cetrotide, Merck Serono Europe Ltd, London, UK; or Fyremadel, Ferring, Malmö, Sweden) were started when the leading follicle reached a mean diameter of 13–14 mm. All patients received GnRH agonist 0.2 mg (Decapeptyl, Ipsen PharmaParis, France) to achieve final oocyte maturation when the mean size of at least two follicles was 18 mm. Oocyte retrieval was performed 36 h later under ultrasound guidance.

       Sample collection

      Following oocyte retrieval, cumulus cells were mechanically stripped from each oocyte using fine needles. Mural luteinized granulosa cells were obtained from pooled follicular aspirate of follicles of at least 14 mm mean diameter, as previously described (
      • Ferrero H.
      • Delgado-Rosas F.
      • Garcia-Pascual C.M.
      • Monterde M.
      • Zimmermann R.C.
      • Simon C.
      • Pellicer A.
      • Gomez R.
      Efficiency and purity provided by the existing methods for the isolation of luteinized granulosa cells: a comparative study.
      ) with minor modifications. Briefly, follicular fluid was slowly layered on a 3:1 Ficoll gradient (Histopaque®-1077, Sigma-Aldrich Merck, Darmstadt, Germany) and centrifuged at 400g for 20 min. Follicular-derived cells were collected from the middle layer and washed twice with phosphate-buffered saline (PBS). Cellular aggregates from both cell lines were dissociated using 0.5 ml of a 0.05% trypsin–EDTA solution at 37°C for 5 min; 4.5 ml of standard culture medium (M-199 supplemented with 10% heat-inactivated fetal bovine serum [FBS] and 100 IU/ml of penicillin/streptomycin) was added to stop trypsin digestion, and the cell suspension was centrifuged at 500g for 5 min. Subsequently, cells were incubated for 2 min at room temperature in the presence of red blood cell lysis buffer (Miltenyi Biotec Inc. Merck, Darmstadt, Germany) according to the manufacturer's instructions. Finally, cumulus cells and purified granulosa cells were washed with PBS and resuspended in standard culture media. All the centrifugations were performed at room temperature. Patient samples were occasionally pooled in order to reach the minimum cell number needed to perform the experiments, but always granulosa cells within the same group of patients (control with control; ARA with ARA). The same procedure was followed, when necessary, with cumulus cells. A scheme with the complete experimental protocol is shown in Supplementary Figure 1.
      When collecting and preparing samples, aliquots of pooled follicular aspirates from each group of patients were centrifuged at 800g for 20 min to remove cells. The supernatant consisted of purified follicular fluid which was sterile-filtered using a 0.22 µm pore size membrane filter (Minisart®; Sartorius Stedim Biotech SA, Madrid, Spain), aliquoted and stored at –80°C.

       Assessment of bioenergetic properties

      Characterization of the bioenergetic properties of mural granulosa cells and cumulus cells was performed using an XF24 Extracellular Flux Analyser (Agilent Technologies, Santa Clara, CA, USA). The analyser performs automatic measurements of the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) in real time, the latter being a proxy for lactate formation. Fresh purified granulosa cells were seeded in XF24-well microplates (Agilent Technologies) at a concentration of 3.5  ×  105 cells/well and 3  ×  105 cells/well for cumulus cells. Cells were maintained for 24 h at 37°C and 5% CO2. One hour prior to OCR and ECAR measurements, the supernatant was carefully removed, wells washed with 1 ml of assay medium and, finally, 500 µl of assay medium were added. The assay medium consisted of XF-DMEM medium supplemented with 2% FBS, 5 mmol/l glucose, 5 mmol/l 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 2 mmol/l glutamine, and 6% follicular fluid (see Results section) pH 7.4. Cells were incubated at 37°C for 1 h in a CO2-free incubator before loading the plate into the analyser. The buffering power of the medium was 0.036 mpH units/pmol H+, which was determined by adding known amounts of HCl to the medium and recording the changes using a pH meter (
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ).
      Control experiments were performed with A549 lung adenocarcinoma cells that were obtained from the American Type Culture Collection (ATCC). Cells were cultured at 37°C in a humidified 5% CO2 atmosphere in Gibco Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated FBS (Gibco, Life Technologies, Paisley, UK ), 2 mmol/l glutamine, and 100 IU/ml of penicillin/streptomycin (Gibco, Life Technologies, Paisley, UK) (ATCC, LGC Ltd, Teddington, UK). A549 cells were seeded in XF24-well microplates at a concentration of 3  ×  104 cells/well and experimental medium was XF-DMEM medium with 2% FBS, 5 mmol/l glucose, 2 mmol/l glutamine, and 5 mmol/l HEPES pH 7.4.
      The experimental protocol to determine the bioenergetics parameters was essentially as described in
      • Brand M.D.
      • Nicholls D.G.
      Assessing mitochondrial dysfunction in cells.
      , which has often been called the ‘mitochondrial stress test’. Basal OCR and ECAR were determined by performing four measurements before the addition of the inhibitors or activators. Subsequently, ATP turnover was assessed from the decrease in oxygen consumption after the addition of the ATPase inhibitor oligomycin (2 µmol/l). The ECAR value after the inhibition of mitochondrial ATP synthesis was considered to be the maximal ECAR. The maximal respiratory capacity and the spare respiratory capacity were established from the increase in the rate of respiration elicited by the uncoupler carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP) after two consecutive additions (0.5 and 0.3 µmol/l). Finally, 1 µmol/l rotenone (complex I inhibitor) and 1 µmol/l antimycin A (complex III inhibitor) were added to quantify the non-mitochondrial respiration. ECAR values were corrected estimating the CO2 contribution to the ECAR signal from the correlation between the decreases in the OCR and the ECAR upon the addition of 30 mmol/l 2-deoxyglucose (2-DOG). In order to normalize OCR and ECAR data to take into account differences in cell content, once the experiments were finished, wells were washed with PBS and the protein concentration in each well was determined by bicinchoninic acid assay using bovine serum albumin as standard. The protein concentration of cumulus cells could not be determined because the PBS wash led to a partial detachment of the cells and, thus, measurements were unreliable.

       Adenine nucleotide measurement

      AMP, ADP and ATP concentrations were determined by reverse-phase high performance liquid chromatography (HPLC) essentially as described in
      • De Korte D.
      • Haverkort W.A.
      • van Gennip A.H.
      • Roos D.
      Nucleotide profiles of normal human blood cells determined by high-performance liquid chromatography.
      . A minimum of 4  ×  106 purified granulosa cells were suspended in 10 ml of the same assay medium described in the previous section and incubated at 37°C for 1 h. Next, they were centrifuged at 8000g for 30 s, homogenized in 660 mmol/l HClO4 plus 10 mmol/l theophylline and kept on ice. The homogenates were centrifuged once more at 16,000g for 15 min at 4°C. The supernatants were neutralized with 2.8 mol/l K3PO4 until a pH between 6 and 7 was reached and stored at –80°C. Having completed the collection, samples were thawed and centrifuged at 16,000g for 15 min at 4°C. The supernatants were passed through a 0.45 µm filter and analysed with a Shimadzu Prominence chromatograph (Canby, Oregon, USA) using a C18 column (Mediterranea SEA18, Teknokroma, Spain). Peaks were identified according to the retention times of standard adenine nucleotides. Chromatograms were analysed using PeakFit software (version 4.12, Systat Software Inc., London, UK). Peak assignment was confirmed using samples treated with 2 µmol/l oligomycin plus 30 mmol/l 2-DOG in which the AMP and ADP peaks markedly increase. The cellular energy charge was calculated using the equation (ATP+ADP/2)/(ATP+ADP+AMP) (
      • Atkinson D.E.
      The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers.
      ).

       Ethical approval

      The Ethics Committee of the University Hospital Puerta de Hierro (Majadahonda, Spain) approved the final version of the study protocol (identification code 1512-MAD-064-JG, 24 September 2018), which also complied with Spanish and European legislation on ART. All participants signed an informed consent.

       Statistical analysis

      Data analysis and graphing were performed using SigmaPlot v14 (Systat Software Inc.) and SPSS v24 (SPSS Inc., Chicago, IL, USA). The continuous variables were reported as mean ± SD or SEM and compared using the t-test or analysis of variance, accordingly. The parameters expressed as medians with ranges presented a non-normal distribution and were compared by the Mann–Whitney U-test. As appropriate, the categorical variables were analysed by either the chi-squared test or Fisher's exact test and described as percentages. Statistical significance was set at a two-tailed P-value of <0.05.

      Results

       Characterization of the bioenergetic profile of human granulosa and cumulus cells

      In order to characterize the bioenergetic properties of both mural luteinized granulosa cells and cumulus cells, a cohort of oocyte donors aged under 35 was used. The characterization was performed using the XF24 Extracellular Flux Analyser. Experiments using this technology ought to be designed in culture media supplemented with a set of substrates in order to understand the specific metabolic requirements of the cells. The more common supplements are glucose, pyruvate or glutamine. Depending on the experimental set-up, cells will display differences in their bioenergetic profile and associated parameters such as the respiratory capacity or the balance between aerobic glycolysis and oxidative phosphorylation (OXPHOS). The standard conditions commonly used to grow both granulosa and cumulus cells include high glucose M-199 media with 10% FBS [34–36]. However, experiments with the XF24 were modified to accommodate standard assay conditions including the use of the Seahorse XF-DMEM medium buffered with 5 mmol/l HEPES and containing 2% FBS and 2 mmol/l glutamine. Additionally, 5 mmol/l glucose was used to approximate normoglycemic concentrations.
      Initial experiments on the XF24 revealed an unusual profile in which the addition of the uncoupler FCCP caused a modest and transient stimulation of respiration that was followed by a decline in the respiratory rate (Figure 1a). The presence of glutamine and/or pyruvate did not alter the profile significantly (data not shown). Inside the pre-ovulatory follicles, cells are immersed in a complex medium, the follicular fluid, containing a variety of metabolites, hormones and other protein factors that are essential for oocyte developmental competence (
      • Dumesic D.A.
      • Meldrum D.R.
      • Katz-Jaffe M.G.
      • Krisher R.L.
      • Schoolcraft W.B.
      Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health.
      ;
      • Fortune J.E.
      Ovarian follicular growth and development in mammals.
      ;
      • O'Gorman A.
      • Wallace M.
      • Cottell E.
      • Gibney M.J.
      • McAuliffe F.M.
      • Wingfield M.
      • Brennan L.
      Metabolic profiling of human follicular fluid identifies potential biomarkers of oocyte developmental competence.
      ). Therefore, the current study tested whether the addition of follicular fluid to the assay medium would improve the respiratory capacity and/or the bioenergetic properties of granulosa and cumulus cells. Interestingly, the addition of low concentrations of follicular fluid (2–6% v/v) markedly increased the response to FCCP in the two cell types (Figure 1b and Supplementary Figure 2). Higher follicular fluid concentrations (10%) compromised the adherence of the cells to the plates. Therefore, the standard medium for the subsequent bioenergetic studies always contained the Seahorse XF-DMEM medium supplemented with 6% follicular fluid, 5 mmol/l glucose, 5 mmol/l HEPES, 2 mmol/l glutamine and 2% FBS. Under these conditions, the stimulation of respiration by FCCP was 107 ± 6.1% for granulosa cells and 146 ± 5.9% for cumulus cells. The rate of glycolysis, as determined from the extracellular acidification rate, reveals the high glycolytic activity of the two cell types. Remarkably, ECAR could not be further stimulated when mitochondrial ATP production was inhibited by oligomycin (Figure 2). This was observed in both cell types and it was not influenced by the presence of follicular fluid. Their glycolytic profile is also reflected in the OCR/ECAR ratio, which was 1.00 ± 0.036 pmol O2/mpH unit for granulosa cells and 2.25 ± 0.12 pmol O2/mpH unit for cumulus cells in the presence of 6% follicular fluid. Although there is a statistically significant difference between the two types of cells (P < 0.001), both values correspond to highly glycolytic cells (
      • Zhang J.
      • Nuebel E.
      • Wisidagama D.R.R.
      • Setoguchi K.
      • Hong J.S.
      • van Horn C.M.
      • Imam S.S.
      • Vergnes L.
      • Malone C.S.
      • Koehler C.M.
      • Teitell M.A.
      Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells.
      ).
      Figure 1
      Figure 1Influence of follicular fluid (FF) on the bioenergetics of luteinized granulosa cells (GC) and cumulus cells (CC). (A) Oxygen consumption rates (OCR) of granulosa cells in the absence (black circles) or in the presence (red circles) of 6% FF in the experimental medium. Additions are indicated with the arrows: ‘Oligo’, 1 µmol/l oligomycin; ‘FCCP’, two consecutive additions of 0.5 and 0.3 µmol/l carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; ‘Rot+AA’, 1 µmol/l rotenone plus 1 µmol/l antimycin A (see Supplementary Figure 2 for further details). (B) Dependence of the maximum respiratory capacity on the presence of FF in GC (red circles) and CC (blue circles). The results are presented as mean ± SEM of six (GC + no FF), four (GC + 2% FF), four (GC + 4% FF), 11 (GC + 6% FF), four (CC + no FF), four (CC + 2% FF), six (CC + 4% FF) and three (CC + 6% FF) independent experiments.
      Figure 2
      Figure 2The rate of glycolysis (extracellular acidification rate, ECAR) in luteinized granulosa (GC) and cumulus cells (CC). (A) Effect of oligomycin on the rate of lactate formation (ECAR) of GC in the presence of 6% follicular fluid (FF). Data points represent the mean ± SEM of six independent experiments. (B) Change in ECAR in response to the addition of oligomycin in GC and CC both in the presence and absence of FF. Data are expressed as the per cent change with respect to basal values. (C) Bioenergetic profile of GC and CC expressed as the OCR/ECAR ratio in the presence and absence of FF. Bars represent the mean ± SEM of 11 (GC + 6% FF), six (GC + no FF), three (CC + 6% FF) and four (CC no FF) independent experiments. ***P < 0.001 between the indicated groups (analysis of variance test).
      The bioenergetic profile of mural granulosa cells and cumulus cells presented an intriguingly high component of non-mitochondrial respiration, which should be due to oxygen-consuming processes like those catalysed by cyclooxygenases, lipoxygenases or NADPH oxidases (NOX). In most cellular systems the non-mitochondrial OCR generally accounts for less than 10% of the basal OCR (
      • Brand M.D.
      • Nicholls D.G.
      Assessing mitochondrial dysfunction in cells.
      ) (the bioenergetic profile of A549 cells is shown in Supplementary Figure 3A for comparison). There are important exceptions, such as the activity of non-mitochondrial NOX in macrophages, which can contribute significantly to cellular oxygen uptake. Under the experimental conditions in this study, the non-mitochondrial respiration in granulosa cells accounted for 56 ± 1.1% of the basal OCR. The NOX inhibitors apocynin and VAS2870 had no effect on the non-mitochondrial respiration of granulosa cells, thus ruling out the involvement of NOX (Supplementary Figure 3B).

       Effects of senescence on the energy metabolism of granulosa cells

      Because female fertility and IVF outcomes dramatically decline with age, a study was designed to explore potential changes in the energy metabolism of luteinized granulosa cells from a group of patients of ARA. The study was restricted to mural granulosa cells because the yield of cells from each patient was significantly higher and, as previously stated, the attachment of the cumulus cells to the XF24 plate was weaker. Patient characteristics, cycle parameters and ovarian response to ovarian stimulation are shown in Table 1. The mean age of the ARA group was 40.8 ± 2.0 while in the control group it was 24.1 ± 3.9. Therefore, the difference between the two experimental groups allowed the effect of age-related infertility on the bioenergetics of human granulosa cells to be explored. The mean concentration of anti-Müllerian hormone (AMH) in the ARA group was 1.27 ± 0.95 ng/ml. AMH concentrations of egg donors are not routinely quantified in the study institution, however the antral follicle count (AFC) was remarkably higher in this group (19.0 ± 4.4 versus 9.0 ± 4.2; P < 0.001). The ARA group required higher gonadotrophin doses [2000 IU (775–4800) versus 2250 IU (1350–4275); P < 0.001]. As expected, the number of oocytes retrieved was significantly higher in the egg donor group [19 (9–54) versus 8 (2–25); P < 0.001], as was the number of mature oocytes [14 (5–41) versus 6 (1–23); P < 0.001)].
      Knowing the influence of the follicular fluid on granulosa cell bioenergetics, the assay media for the control and ARA patients were supplemented with follicular fluid isolated from individuals belonging to their respective groups. It might be expected that the observed bioenergetic properties could be conditioned by the cells, the composition of the follicular fluid or a combination of the two factors. Figure 3 shows a summary of the bioenergetic properties of granulosa cells from the control donors and the ARA group. It is apparent that the basal rate of respiration and the ECAR are significantly reduced in the ARA group, thus indicating a global decrease in the energy metabolism as women age (Figure 3a, *P = 0.046 and Figure 3b, *P = 0.026, **P = 0.009, between the indicated groups). As expected, the parallel decrease in the two parameters does not modify the bioenergetic profile as estimated by the OCR/ECAR ratio (Figure 3c). Notably, the decrease in the basal respiration in the ARA group is not reflected in the maximal respiratory capacity of the cells, which remains unchanged (Figure 3d).
      Figure 3
      Figure 3Bioenergetic parameters of mural luteinized granulosa cells (GC) from young donors (empty bars) and advanced reproductive age (ARA) women (grey bars) and influence of the follicular fluid (FF) origin: DFF = FF from donors; AFF = FF from ARA group. (A) Basal rate of mitochondrial respiration. OCR = oxygen consumption rate. (B) Basal rate of glycolysis as determined from the rate of lactate formation (extracellular acidification rate, ECAR) value. (C) Bioenergetic profile expressed as the OCR/ECAR ratio. (D) Maximum respiratory capacity. Bars represent the mean ± SEM of 11 (donor DFF and ARA DFF) or nine (donor AFF, ARA AFF) independent experiments. A, *P = 0.046 and B, *P = 0.026, **P = 0.009, between the indicated groups (analysis of variance test).
      The influence of the origin of the follicular fluid used to supplement the media was investigated by performing experiments in which the donor follicular fluid was used to supplement the media for the ARA group and vice versa. Figure 3 reveals that the highest rates of respiration and glycolysis were observed in the donor group with medium supplemented with the donor follicular fluid. The remaining combinations led to a decrease in the energy metabolism although under no conditions was the maximal respiratory capacity affected.

       Adenine nucleotide concentrations in human luteinized granulosa cells

      To investigate whether the decreased metabolism is due to senescence and causes a reduction in the cellular energy charge, the adenine nucleotide concentrations (ATP, ADP and AMP) of the granulosa cells in the two experimental groups were determined. The incubation media was the same as in the XF24 experiments, supplementing the XF-DMEM medium with 6% follicular fluid of the respective experimental group. Figure 4 summarizes the changes in the adenine nucleotide pool and reveals that granulosa cells from the ARA group exhibited a marked decrease in the cellular energy charge (P < 0.001), which is also reflected in the ATP/ADP (P < 0.001) and ATP/AMP ratios (P < 0.001). Because experiments on the XF24 analyser had shown that inhibition of mitochondrial ATP synthesis did not cause a compensatory increase in glycolysis (ECAR), the effect of oligomycin on the adenine nucleotide pool was studied. Results in Figure 4 (hatched bars) show that this inhibitor markedly decreased ATP concentrations in both groups (P < 0.001) confirming that, because glycolysis cannot be further stimulated, it cannot compensate for the loss of mitochondrial ATP synthesis. As expected, the combination of oligomycin and 2-DOG totally collapsed the energy levels.
      Figure 4
      Figure 4Effect of reproductive senescence on the adenine nucleotide pool in mural luteinized granulosa cells (GC) in the presence of 6% follicular fluid (FF). Empty bars, control donors. Grey bars, ARA group. Hatched bars in each group are the values obtained in the presence of oligomycin. Black bar is the value in the presence of oligomycin and 2-deoxyglucose (O+D). (A) Energy charge calculated using the equation (ATP+ADP/2)/(ATP+ADP+AMP). (B) ATP/ADP ratio. (C) ATP/AMP ratio. Bars represent the mean ± SEM of 14 (donor), nine (ARA), seven (donor + oligomycin), five (ARA + oligomycin), seven (donor + oligomycin + deoxyglucose) independent determinations. ***P < 0.001 between the indicated groups (analysis of variance test).

      Discussion

      The analysis of the bioenergetic properties of human mural luteinized granulosa cells and cumulus cells presented here has allowed further understanding of the metabolism of these two follicular cell types, which are known to influence the maturation of the oocyte and, ultimately, IVF outcomes (
      • Liu Y.
      • Han M.
      • Li X.
      • Wang H.
      • Ma M.
      • Zhang S.
      • Guo Y.
      • Wang S.
      • Wang Y.
      • Duan N.
      • Xu. B.
      • Yin J.
      • Yao Y.
      Age-related changes in the mitochondria of human mural granulosa cells.
      ;
      • Shufaro Y.
      • Lebovich M.
      • Aizenman E.
      • Miller C.
      • Simon A.
      • Laufer N.
      • Saada A.
      Human granulosa luteal cell oxidative phosphorylation function is not affected by age or ovarian response.
      ). These cells are believed to be potential biomarkers of oocyte quality (
      • Anderson S.H.
      • Glassner M.J.
      • Melnikov A.
      • Friedman G.
      • Orynbayeva Z.
      Respirometric reserve capacity of cumulus cell mitochondria correlates with oocyte maturity.
      ;
      • Dumesic D.A.
      • Meldrum D.R.
      • Katz-Jaffe M.G.
      • Krisher R.L.
      • Schoolcraft W.B.
      Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health.
      ), and it has been proposed that alterations in their energy metabolism could lead to infertility (
      • Dong Z.
      • Huang M.
      • Liu Z.
      • Xie P.
      • Dong Y.
      • Wu X.
      • Qu Z.
      • Shen B.
      • Huang X.
      • Zhang T.
      • Li J.
      • Liu J.
      • Yanase T.
      • Zhou C.
      • Xu Y.
      Focused screening of mitochondrial metabolism reveals a crucial role for a tumor suppressor Hbp1 in ovarian reserve.
      ;
      • Hsu A.L.
      • Townsend P.M.
      • Oehninger S.
      • Castora F.J.
      Endometriosis may be associated with mitochondrial dysfunction in cumulus cells from subjects undergoing in vitro fertilization-intracytoplasmic sperm injection, as reflected by decreased adenosine triphosphate production.
      ;
      • Liu Y.
      • Han M.
      • Li X.
      • Wang H.
      • Ma M.
      • Zhang S.
      • Guo Y.
      • Wang S.
      • Wang Y.
      • Duan N.
      • Xu. B.
      • Yin J.
      • Yao Y.
      Age-related changes in the mitochondria of human mural granulosa cells.
      ;
      • Shufaro Y.
      • Lebovich M.
      • Aizenman E.
      • Miller C.
      • Simon A.
      • Laufer N.
      • Saada A.
      Human granulosa luteal cell oxidative phosphorylation function is not affected by age or ovarian response.
      ). The results of this study show (i) the high glycolytic profile of these two cell types, (ii) that the respiratory capacity relies on the presence of follicular fluid in the experimental media, and (iii) that luteinized granulosa cells from older women present lower respiration and aerobic glycolysis, leading to a decrease in cellular ATP concentrations. This altered energy metabolism could provide a mechanistic explanation for the decrease in reproductive potential when women age.
      The follicular fluid is a complex medium produced by granulosa and thecal cells, and is also enriched with biomolecules from plasma, which influences oocyte competence and quality (
      • Dumesic D.A.
      • Meldrum D.R.
      • Katz-Jaffe M.G.
      • Krisher R.L.
      • Schoolcraft W.B.
      Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health.
      ;
      • Freitas C.
      • Neto A.C.
      • Matos L.
      • Silva E.
      • Ribeiro Â.
      • Silva-Carvalho J.L.
      • Almeida H.
      Follicular fluid redox involvement for ovarian follicle growth.
      ;
      • Hennet M.L.
      • Combelles C.M.H.
      The antral follicle: a microenvironment for oocyte differentiation.
      ). The main components are steroid hormones, metabolites (amino acids, glucose, fatty acids, etc.), polysaccharides, proteins and growth factors, among others (
      • Dumesic D.A.
      • Meldrum D.R.
      • Katz-Jaffe M.G.
      • Krisher R.L.
      • Schoolcraft W.B.
      Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health.
      ;
      • Fortune J.E.
      Ovarian follicular growth and development in mammals.
      ;
      • O'Gorman A.
      • Wallace M.
      • Cottell E.
      • Gibney M.J.
      • McAuliffe F.M.
      • Wingfield M.
      • Brennan L.
      Metabolic profiling of human follicular fluid identifies potential biomarkers of oocyte developmental competence.
      ). The presence of enzymatic and non-enzymatic antioxidants is essential to maintain homeostatic concentrations of reactive oxygen species that are potentially harmful but also necessary for oocyte growth (Da
      • Da Broi M.G.
      • Giorgi V.S.I.
      • Wang F.
      • Keefe D.L.
      • Albertini D.
      • Navarro P.A.
      Influence of follicular fluid and cumulus cells on oocyte quality: clinical implications.
      ;
      • Freitas C.
      • Neto A.C.
      • Matos L.
      • Silva E.
      • Ribeiro Â.
      • Silva-Carvalho J.L.
      • Almeida H.
      Follicular fluid redox involvement for ovarian follicle growth.
      ). Although the current data have revealed that the follicular fluid is a fundamental element to attain the full respiratory capacity of mural granulosa cells and cumulus cells, it was beyond the scope of the current work to elucidate which are the critical components. The respiratory capacity of the cells was determined by adding the uncoupling agent FCCP and, therefore, its physiological relevance should be interpreted with caution. However, if this bioenergetic parameter reflects the OXPHOS capacity of the oocyte and/or the supporting cells, then follicular fluid components could significantly affect the outcome of the energy-demanding processes that take place upon fertilization.
      The other pathway that provides energy to the cell is glycolysis. The current data show that, under these experimental conditions, the rate of glycolysis appears to be at its maximum because the inhibition of mitochondrial ATP synthesis did not result in a compensatory increase in glycolysis (Figure 2) and, consequently, inhibition of mitochondrial ATP synthesis led to a collapse in the cellular energy charge (Figure 4). These findings emphasize the importance of the two pathways in maintaining an adequate ATP supply and sustaining development. The decrease in both OXPHOS and glycolysis could be the result of a decreased energy demand of the cell due to reproductive senescence. If this were to be the case, the cellular energy charge should remain unchanged. However, the data from this study clearly show that ageing impairs cellular bioenergetics (Figure 3) and as a result there is a significant decrease in ATP concentrations (Figure 4). It is puzzling that despite the lower energy charge observed in the ARA group, OXPHOS is not increased to attempt to restore the optimal ATP concentrations despite the existence of spare respiratory capacity. In this context, it must be pointed out that the maximal respiratory capacity remained unchanged in the two experimental groups. This is important because it reveals that the mitochondrial mass (total respiratory capacity) does not appear to be affected by ageing. A recent study that used flow cytometry to evaluate mitochondrial mass and respiratory capacity of cumulus cells failed to detect significant differences between patients younger and older than 35 years of age (
      • Anderson S.H.
      • Glassner M.J.
      • Melnikov A.
      • Friedman G.
      • Orynbayeva Z.
      Respirometric reserve capacity of cumulus cell mitochondria correlates with oocyte maturity.
      ). Therefore, the molecular basis for the diminished energy metabolism cannot currently be envisaged. However, there are reports showing, for example, that in human granulosa cells deficiencies in complex V (
      • Liu Y.
      • Han M.
      • Li X.
      • Wang H.
      • Ma M.
      • Zhang S.
      • Guo Y.
      • Wang S.
      • Wang Y.
      • Duan N.
      • Xu. B.
      • Yin J.
      • Yao Y.
      Age-related changes in the mitochondria of human mural granulosa cells.
      ) or variations in sirtuin expression (
      • Tatone C.
      • Di Emidio G.
      • Barbonetti A.
      • Carta G.
      • Luciano A.M.
      • Falone S.
      • Amicarelli F.
      Sirtuins in gamete biology and reproductive physiology: emerging roles and therapeutic potential in female and male infertility.
      ) could be behind mitochondrial dysfunction in ovarian ageing.
      Compelling evidence suggests that the composition of the human follicular fluid plays a role in IVF outcomes (
      • Cordeiro. F.B.
      • Montani D.A.
      • Pilau E.J.
      • Gozzo F.C.
      • Fraietta R.
      • Lo Turco E.G.
      Ovarian environment aging: follicular fluid lipidomic and related metabolic pathways.
      ;
      • O'Gorman A.
      • Wallace M.
      • Cottell E.
      • Gibney M.J.
      • McAuliffe F.M.
      • Wingfield M.
      • Brennan L.
      Metabolic profiling of human follicular fluid identifies potential biomarkers of oocyte developmental competence.
      ;
      • Wen X.
      • Kuang Y.
      • Zhou L.
      • Yu B.
      • Chen Q.
      • Fu Y.
      • Yan Z.
      • Guo H.
      • Lyu Q.
      • Xie J.
      • Chai W..
      Lipidomic components alterations of human follicular fluid reveal the relevance of improving clinical outcomes in women using progestin-primed ovarian stimulation compared to short-term protocol.
      ); however, the defects in energy metabolism during reproductive senescence are only partially explained by its composition. Thus, basal rates of respiration and glycolysis in granulosa cells from the control group were significantly reduced in the presence of follicular fluid from older women, but follicular fluid from the young donors failed to improve the bioenergetic parameters in the ARA group (Figure 3). Recently, a porcine in-vitro maturation model showed that supplementing the maturation medium with follicular fluid not only increased mitochondrial DNA copy number and the ATP content in both oocytes and cumulus cells, but also the survival of cumulus cells and the blastulation rate (
      • Ogawa K.
      • Itami N.
      • Ueda M.
      • Kansaku K.
      • Shirasuna K.
      • Kuwayama T.
      • Iwata H.
      Non-esterified fatty acid-associated ability of follicular fluid to support porcine oocyte maturation and development.
      ).
      In conclusion, although many reports have pointed to mitochondria playing a critical role in oocyte quality and IVF outcome (
      • Cecchino G.N.
      • Seli E.
      • Alves da Motta E.L.
      • García-Velasco J.A.
      The role of mitochondrial activity in female fertility and assisted reproductive technologies: overview and current insights.
      ;
      • Demain L.A.M.
      • Conway G.S.
      • Newman W.G.
      Genetics of mitochondrial dysfunction and infertility.
      ;
      • Kasapoglu I.
      • Seli E.
      Mitochondrial dysfunction and ovarian aging.
      ;
      • May-Panloup P.
      • Chretien M.F.
      • Malthiery Y.
      • Reynier P.
      Mitochondrial DNA in the oocyte and the developing embryo.
      ), this study shows for the first time that both OXPHOS and glycolysis are decreased in mural luteinized granulosa cells during reproductive ageing and that these two concerted events lead to a decrease in ATP concentrations. Moreover, the current data reveal that mitochondrial mass does not decrease with ageing because mural granulosa cells retain their full respiratory capacity. It must be pointed out that all samples were obtained from women that had gone through stringent exclusion criteria to ensure there were no underlying pathologies that could influence fertility. Therefore, it seems reasonable to conclude that the observed deficiencies in energy metabolism are related to intrinsic functional properties due to reproductive ageing that are likely to influence overall IVF performance. A new window of opportunity for diagnostic and therapeutic tools may arise from studies focusing on the bioenergetics of granulosa cells, oocytes and embryos.

      Data availability statement

      The data underlying this article will be shared on reasonable request to the corresponding author.

      Acknowledgements

      This work was supported by IVI-Madrid. GNC received grants from the Capes Foundation, Brazil/PDSE/process number 88881.132905/2016-01.

      Appendix. Supplementary materials

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      Biography

      Gustavo Cecchino is a fertility specialist at the Mater Prime Clinic (Brazil). He has completed a two-year fellowship programme at IVI-Madrid (Spain) and is currently finishing his PhD in ovarian ageing and energy metabolism. His current research interests include mitochondrial bioenergetics in reproductive ageing and new technologies to improve reproductive outcomes.
      Key message
      Luteinized granulosa cells of women of advanced reproductive age show lower respiration and aerobic glycolysis, which lead to a decrease in cellular ATP levels. The observed reduction in energy metabolism is likely to influence female reproductive potential.