Mitochondrial FIS1 level in cumulus cells correlates with morphological grades of human cleavage-stage embryos

doi:10.21203/rs.3.rs-5298954/v1

Background

Advanced-age women have a lower good-quality embryo rate (GQER) compared to young women. However, GQER varies widely within the same age group, suggesting that factors beyond age influence embryo quality. Mitochondria regulate the metabolism of their host cells through dynamic fission and fusion alterations. Specifically, cumulus cell (CC) mitochondria regulate not only the metabolism of CCs but also of adjacent oocytes. This study aims to investigate the relationship between CC mitochondrial dynamics and oocyte developmental potential post-fertilization.

Methods

CCs were collected from 183 women aged 25–45 undergoing single sperm intracytoplasmic injection-embryo transfer treatments. Samples were stratified by age into young (< 35) and advanced-age (≥ 35) groups. Each group was further subdivided into high and low subgroups based on the Day 3 GQER. Mitochondrial morphology, dynamics, and fission-fusion gene expression were compared among groups and subgroups.

Results

Consistent with the literature, data analysis from our laboratory revealed significant variances in GQER among individuals of the same age group. Morphological analysis suggested a negative correlation between GQER and mitochondrial length in CCs (P < 0.0001, r=-0.38). Live-cell imaging showed that both fission and fusion frequencies of CC mitochondria in the advanced-age group were lower than those in the young group (P = 0.009, P = 0.01). Additionally, within the advanced-age group, CC mitochondria from the low GQER subgroup exhibited lower fission frequency and fission-fusion ratios compared to the high GQER subgroup (P = 0.04, P = 0.01). Consequently, GQER positively correlated with mitochondrial fission-fusion ratio in CCs (P = 0.01, r = 0.44). Notably, there were no significant differences in the expression of mitochondrial fusion-related proteins (OPA1, MFN1, and MFN2) between the advanced-age and young groups or among the subgroups. However, levels of fission proteins, including FIS1 and MFF, were significantly lower in the advanced-age group compared to the young group and in the low GQER subgroup compared to their high GQER counterparts. qPCR results further indicated that fis1 and mff mRNA levels in CCs were positively correlated with GQER (P < 0.0001, r = 0.55; P = 0.0025, r = 0.41).

Conclusions

Mitochondrial morphology, dynamics, and fission-fusion gene expression in CCs influence early embryonic development, independent of age. Of these factors, the FIS1 level shows the most robust correlation with GQER.

Mitochondrial dynamics

cumulus cells

preimplantation embryonic development

maternal age

In recent years, there has been a growing tension between delayed childbearing and age-associated fertility decline [1–3]. While assisted reproductive technologies (ART) provide a degree of fertility enhancement, significant individual differences in embryonic development potential persist among women of advanced maternal age [4], suggesting that factors beyond age influence embryo quality. The limited effectiveness of ART for advanced-age women primarily stems from the oocyte's developmental potential [5], which is established during prolonged follicular development through complex interactions with surrounding somatic cells (theca, mural granulosa, and cumulus granulosa cells) [6]. ART interventions, focusing on oocytes that have completed this critical process, are thus inherently restricted in their capacity to mitigate age-related fertility decline. Accumulating evidence demonstrates oocytes' inability to synthesize pyruvate and cholesterol or uptake alanine, necessitating reliance on granulosa cells, particularly cumulus cells (CCs), for intermediates essential to their primary metabolic processes [7]. The intimate relationship between CCs and oocyte establishes CCs as an optimal non-invasive biomarker for assessing oocyte quality and embryonic developmental potential, providing significant prognostic value in clinical settings.

Previous studies suggested that CCs in women of advanced maternal age exhibited significantly reduced nutritional support capacity compared to those in young women [8, 9]. Mitochondrial dysfunction in CCs has been identified as a pivotal factor in this decline [10–12]. Mitochondria, unique as cellular organelles bearing genetic material, undergo a distinct lifecycle encompassing biogenesis, dynamics, and quality control [13]. Environmental perturbations can impact mitochondrial lifecycle, prompting cellular physiological adaptations. However, the specific aspect of mitochondrial lifecycle in CCs primarily responsible for dysfunction in advanced maternal age remains elusive [14, 15]. Recent research has revealed that mitochondrial morphology correlates with differential metabolic capacities for carbohydrates, lipids, and proteins. For instance, elongated mitochondria preferentially metabolize fatty acids, while shorter mitochondria primarily facilitate glucose metabolism [16, 17]. Mitochondrial morphology is modulated through ongoing fission and fusion events to meet cellular adaptive requirements [18]. Considering the metabolic support function of CCs for oocytes, we posit that mitochondrial dynamics imbalance may be the critical factor underlying the marked decline in CC nutritional support capacity in women of advanced maternal age.

The balance of mitochondrial dynamics in cells is strictly regulated by complex molecular machineries. Fusion machinery involves three GTPases: mitochondrial fusion proteins (MFN1 and MFN2) located on the outer membrane and optic atrophy protein 1 (OPA1) located on the inner membrane [18]. Fission is mediated by dynamin-related protein 1 (DRP1) and its two adapters, mitochondrial fission factor (MFF) and mitochondrial fission protein 1 (FIS1) [19]. Among them, MFF governs midzone fission to generate new mitochondria, while FIS1 directs peripheral fission to remove depolarized mitochondria for subsequent mitophagy [20]. To test our hypothesis, we conducted a comparative study between the advanced-age and young groups. We collected CCs from both groups and assessed early embryonic developmental potential. The investigation encompassed analyses of mitochondrial morphology, dynamics characteristics, and expression levels of regulatory genes across age groups and their respective high and low good-quality embryo rate (GQER) subgroups (Fig. 1). We examined the correlation between mitochondrial dynamics and early embryonic quality, confirming an association between fission-fusion imbalance and diminished embryonic quality. This study establishes a novel link between CC mitochondrial dynamics and early embryonic quality, potentially offering new insights into improving fertility preservation strategies for women of advanced maternal age.

Study participants

This study enrolled 183 women who received assisted reproductive treatment at the Reproductive Medicine Center of Zhongshan Hospital between June 2023 and April 2024. Eligible women were aged from 25 to 45 years old, undergoing ICSI cycles with an indication of tubal, male, or unexplained infertility, after controlled ovarian hyperstimulation with gonadotropin releasing hormone (GnRH) antagonist. Exclusion criteria were diagnosis of polycystic ovary syndrome, premature ovarian failure, chromosomal abnormalities, thyroid disease, or other endocrine disorders. Additionally, women were excluded if their partners were diagnosed with severe oligozoospermia (sperm concentration < 5 × 10⁶ sperm per mL) or asthenozoospermia (sperm progressive motility < 10%). The study has received approval from the Ethics Committee of Zhongshan Hospital Fudan University (B2024-185R), and all participants have provided informed consent.

Cumulus cells collection and culture

Cumulus-oocyte complexes (COCs) were obtained through follicle puncture. Only COCs from follicles with diameters exceeding 15 mm were included to ensure that all CC samples were derived from mature oocytes. After oocyte retrieval, CCs were denudated from the oocyte using hyaluronidase (Vitrolife, Sweden). Discarded CCs were subsequently collected in a 1.5 mL microcentrifuge tube and centrifuged at 400g for 5 minutes. The cell pellet was then resuspended in culture medium for further culture or otherwise was washed by PBS for molecular biological analyses. For primary CCs culture, the cell pellet was resuspended in the IVM medium and cultured on a 35 mm glass-bottom dish (Cellvis, CA, USA) in a 37°C incubator with 5% CO₂. The IVM medium contained tissue culture medium 199 supplemented with 0.22 mM pyruvic acid, 0.075 IU/ml FSH, 0.5 IU/ml hCG, 0.1 mg/ml 17β-estradiol, 0.6 g/l penicillin and streptomycin, in addition to 20% (v/v) FBS. All utilized reagents were procured from Sigma Chemical Co.

Embryo quality assessment

For every participant, their embryos were evaluated at the cleavage stage on Day 3 and at the blastocyst stage on Day 5/6. Since not all embryos underwent blastocyst culture, the Day 3 embryo grading was primarily used for assessing embryonic quality. A team of experienced embryologists evaluated each embryo based on the morphological criteria proposed by Veeck [21]. The evaluation goes as follows. Grade I: Blastomeres of equal size with no cytoplasmic fragmentation; Grade II: Blastomeres of equal size with minor cytoplasmic fragmentation or blebs; Grade III: Blastomeres of distinctly unequal size with little to no cytoplasmic fragmentation; Grade IV: Blastomeres of equal or unequal size with significant cytoplasmic fragmentation; Grade V: Few blastomeres of any size with severe or complete fragmentation. Good-quality embryos were defined as those containing 7–9 cells and classified as Grade I or II. For every participant, their individual GQER was defined as the number of good-quality embryos divided by the number of cleavage stage embryos.

MitoTracker staining

To perform live cell tracing, MitoTracker™ Red FM Dye (Invitrogen, CA, USA) and NucBlue™ Live ReadyProbes™ (Invitrogen) were diluted into aforementioned complete culture medium at ratio of 1:2000 and 1:1000, respectively, for mitochondrial staining and nucleus labelling. Culture medium of CCs was then replaced with the staining solution and incubated for 20 minutes at 37°C with 5% CO₂. Subsequently, staining solution was washed off twice with fresh complete culture medium, and the cells were further cultured for an additional 30 minutes to remove any residual dye.

Live cell imaging

After MitoTracker staining, CCs were transferred to a temperature-controlled cage incubator (Okolab, Italy) set to 37℃ with 5% CO₂. Images were acquired by confocal microscopy and super-resolution imaging. For confocal microscopy, we employed a Nikon C2 Si Confocal Microscope (Nikon, Japan). Super-resolution imaging was performed using High Intelligent and Sensitivity Structured Illumination Microscopy (HIS-SIM), a technology provided by Guangzhou Computational Super-resolution Biotech Co. For morphological observations, three random fields of view were captured for each sample using the 60× Nikon CFI Apo TIRF Oil objective (Nikon). To track mitochondrial motion, 10 random fields of view were selected within each sample and recorded as time-lapse images at 5-second intervals in a total duration of 5 minutes.

Image processing and analysis

Morphological characterization of mitochondria was performed on the Mitochondria-Analyzer plugin in ImageJ software [22]. For each sample, three images were processed through binarization to distinguish the true fluorescent signal from the background signal. The binarized images were then analyzed to extract the morphological characteristics of individual mitochondria. One measure taken to assess the mitochondrial shape was the Form Factor, with size calculated as \(\:{p}^{2}/\left(4\pi\:a\right)\) where \(\:p\) represents the length of mitochondrial boundary and \(\:a\) represents the total surface area. A Form Factor value of 1 indicates a round shape, while higher values indicate more elongated and irregular shapes. Mitochondria were categorized based on their Form Factor values: spheres (1-1.45), short tubules (1.46-2.0), and elongated tubules (> 2.0).

Mitochondrial tracking and determination of fission and fusion events based on time-lapse images were performed by the Mitometer tool in MATLAB R2020a [23]. Firstly, background removal was applied to obtain segregated images. Subsequently, the software visualized all mitochondrial tracks where confident tracks were selected for further analysis. Fission and fusion events were then calculated based on the identified tracks. For each CC sample, time-lapse images from 10 fields of view were processed by the Mitometer. The average fission and fusion events from these fields were calculated for subsequent statistical analysis.

RNA extraction and qRT-PCR analysis

Total RNA was extracted from isolated CCs using Direct-zol™ RNA Microprep Kit (Zymo Research, CA, USA). Concentration of RNA was determined using the Nanodrop (Thermo Fisher Scientific, MA, USA). Reverse transcription was performed on 500 ng of total RNA with the PrimeScript™ RT Master Mix kit (Takara Bio, Japan). qRT-PCR was carried out on a LightCycler 480 II instrument (Roche, Switzerland) using forward and reverse primers at a final concentration of 0.2 µM mixed with the Hieff qPCR SYBR Green Master Mix kit (Yeasen Biotechnology, China). The primer sequences are provided in Supplemental Table 1. The expression of target gene was normalized to the expression level of gapdh and calculated by the 2^−ΔΔCT method.

Western blotting

CCs were lysed by ice-cold RIPA lysis buffer (Beyotime, China) supplemented with Protease Inhibitor Cocktail (Bimake, USA) for 10 minutes. Protein concentration was determined using the BCA Protein Assay Kit (Beyotime), and the loading volume of each sample was adjusted accordingly. The protein samples were then mixed with SDS-PAGE loading buffer (Fude Bio, China) and heated at 99℃ for 10 minutes. Subsequently, the proteins were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, USA).

After blocked with 5% milk at room temperature for 60 minutes, the PVDF membrane was then incubated overnight at 4°C with primary antibodies diluted in 5% milk. The primary antibodies used were anti-MFF (84580, CST, MA, USA), anti-FIS1 (HPA017430, Sigma), anti-DRP1 (8570, CST), anti-MFN1 (14739, CST), anti-MFN2 (9482, CST), anti-OPA1 (157457, Abcam, MA, USA) and anti-β-actin (81115-1-RR, Proteintech, China). After three washes with TBST (Tris-buffered saline with Tween 20), each lasting for 15 minutes, the membrane was incubated with a horseradish peroxidase-conjugated secondary anti-rabbit antibody (7074, CST) (diluted 1:3000 in TBST) at room temperature for one hour. Following three washes with TBST, the membrane was incubated with Tanon™ ECL substrate (Tanon, China) for two minutes, and the chemiluminescent signal was captured using an e-Blot chemiluminescence imager. The gray value of the immunoblotting strips was analyzed by ImageJ software.

Statistical analysis

All statistical analysis were conducted using GraphPad Prism 9. The two-sided Student's t-test was employed to compare continuous variables between two groups that were subject to a normal distribution. For variables that were not normal distributed, the Mann-Whitney U test was utilized to compare the two independent groups. Pearson test or Spearmen test was used to analyze the correlation between the two variables according to data distribution. Fisher's exact test was employed to compare proportions between two groups. Statistical significance was defined as P values < 0.05.

Study population, clinical characteristics, and embryology laboratory outcomes

The study included 183 participants, who were divided into the young group (< 35 years, n = 92) and the advanced-age group (≥ 35 years, n = 91). Clinical characteristics and embryology laboratory outcomes are summarized in Table 1. No statistically significant differences were observed between the groups with respect to body mass index, type of infertility, or baseline serum hormone levels, including luteinizing hormone, follicle-stimulating hormone, estradiol, and progesterone. However, the advanced-age group exhibited significantly lower anti-Müllerian hormone levels and antral follicle counts. In accordance with the existing literature, women of advanced maternal age demonstrated diminished pre-implantation embryo developmental potential compared to their younger counterparts. This was evidenced by significant reductions in retrieved oocytes, good-quality embryos, GQER, blastocysts, and blastocyst formation rates in the advanced-age group. However, our data also revealed some exceptions where some young women exhibited low GQER while some advanced-age women exhibited high GQER. This suggests that while age is a significant factor in determining embryo quality, it is not the only influencing factor.

Table 1

Characteristics of patients.
	< 35 years	≥ 35 years	P value
Number	92	91
Age (years)	30.75 ± 2.46	39.38 ± 3.25	< 0.0001
BMI (kg/m²)	21.98 ± 2.20	22.51 ± 2.77	0.31
Infertility diagnosis
Tubal factor	54 (58.70%)	56 (61.54%)	0.76
Male factor	15 (16.30%)	9 (9.89%)	0.27
Combined factors	18 (19.57%)	20 (21.98%)	0.49
Unexplained	5 (5.43%)	6 (6.59%)	0.72
Basal LH (mIU/mL)	5.74 ± 1.70	6.04 ± 2.46	0.81
Basal FSH (mIU/mL)	6.85 ± 1.79	7.41 ± 2.22	0.08
Basal E2 (pmol/L)	116.40 ± 36.96	124.10 ± 48.83	0.37
Basal P4 (nmol/L)	0.86 ± 0.46	0.80 ± 0.51	0.23
AMH (ng/mL)	2.77 ± 0.93	1.49 ± 1.08	< 0.0001
AFC	15.29 ± 5.66	9.95 ± 4.92	< 0.0001
Number of oocytes retrieved	15.03 ± 6.52	9.99 ± 6.80	< 0.0001
MII oocytes (%)	77.73 ± 16.74	79.34 ± 17.60	0.53
2PN (%)	79.63 ± 14.63	79.58 ± 20.11	0.51
Number of good-quality embryos	4.13 ± 3.15	3.23 ± 2.75	0.04
GQER (%)	43.68 ± 26.98	35.05 ± 25.23	0.04
Number of blastocysts	3.13 ± 3.13	2.32 ± 2.80	0.03
Blastocyst formation rate (%)	40.18 ± 29.43	31.76 ± 33.69	0.04
BMI: body mass index; LH: luteinizing hormone; FSH: follicle-stimulating hormone; E2: estradiol; P4: progesterone; AMH: anti-mullerian hormone; AFC: antral follicle count; PN: pronucleus; GQER: good-quality embryo rate. Data are shown as mean ± SD.

Given the observed intra-group variability in GQER, we conducted a subgroup analysis of patient characteristics. Data from our center, corroborated by multiple studies [24–26], suggest that the average GQER among women of reproductive age typically ranges from 40–50%. Using this benchmark, we stratified the participants into two subgroups: those with a high GQER (≥ 40%) and those with a low GQER (< 40%). No statistically significant differences in clinical parameters were observed between the subgroups (Fig. 2. A-C). In both young and advanced-age groups, laboratory outcomes, including normal fertilization rates, GQER and blastocyst formation rates, were significantly elevated in the high GQER subgroup compared to the low GQER subgroup (Fig. 2. D). These findings suggest that factors beyond age influence embryonic quality. Considering the crucial role of mitochondrial dynamics in cellular metabolism regulation and the essential metabolic support that CCs provide to oocytes, we hypothesized that disruption of the mitochondrial dynamic equilibrium of CCs may contribute to embryo quality decline, independent of maternal age. Consequently, we investigated the relationship between mitochondrial morphology, fission-fusion balance, and gene expression profiles in relation to early embryonic developmental potential.

Abnormal mitochondrial morphology in CCs linked to compromised embryo cleavage

We initially investigated the differences in mitochondrial morphology between the advanced-age and young groups. Using the fluorescent probe MitoTracker^red, we labeled 60 CC samples from the advanced-age group and 51 from the young group, observing them under a confocal microscope equipped with a live-cell imaging system. In the young group, mitochondria were predominantly spherical or short rod-shaped, with a minority showing elongated and hyperfused forms (Fig. 3A). In contrast, the advanced-age group primarily displayed elongated and hyperfused mitochondrial patterns (Fig. 3B). Notably, across both the young and advanced-age groups, mitochondria belonging to the high GQER subgroup largely maintained spherical or short rod-shaped forms. Conversely, those associated with the low GQER subgroup predominantly manifested elongated and hyperfused morphologies (Fig. 3A and B). Statistical analysis revealed that the average mitochondrial length in the advanced-age group was significantly higher than the young group (1.47 vs 1.32 µm, P = 0.02) (Fig. 3C). Further subgroup analysis demonstrated that the average mitochondrial length in the low GQER subgroup was significantly longer than the high GQER subgroup for both age groups (1.41 vs 1.29 µm, P = 0.04; 1.65 vs 1.33 µm, P = 0.003) (Fig. 3C). Correlation analysis indicated a negative relationship between participants' GQER and the average mitochondrial length in their CCs (P < 0.0001, r = -0.38) (Fig. 3D).

We also quantified mitochondria of different shapes (spheres, short tubules, and elongated tubules). Compared to the young group, the advanced-age group exhibited a lower proportion of spherical mitochondria (P = 0.03), no significant difference in short tubular mitochondria, and a higher proportion of elongated tubular mitochondria (P = 0.049) (Fig. 3E). Similarly, compared to the high GQER subgroup, the low GQER subgroup showed a lower proportion of spherical mitochondria (P = 0.04; P = 0.0002), no significant difference in short tubular mitochondria, and a higher proportion of elongated tubular mitochondria (P = 0.028; P = 0.0001) (Fig. 3E). Correlation analysis revealed that the GQER was positively correlated with the proportion of spherical mitochondria (P < 0.0001, r = 0.43), not correlated with the proportion of short tubular mitochondria, and negatively correlated with the proportion of elongated tubular mitochondria (P < 0.0001, r = -0.47) (Fig. 3F-H). These results suggest that the elongation of mitochondria in CCs is associated with decreased early embryonic developmental potential.

Mitochondrial fission-fusion imbalance in CCs associated with reduced GQER

To elucidate the underlying causes of the observed changes in mitochondrial length, we investigated the dynamic characteristics of mitochondrial fission and fusion in 17 CC samples from the advanced-age group and 13 from the young group. For each sample, we captured 10 fields of view, recording each field for five minutes. In CCs from a 29-year-old woman, we observed active mitochondrial movement. Within a 34×34 µm field of view, four fission and two fusion events were captured over a three-minute period (Fig. 4A, Video 1). Conversely, in CCs from a 42-year-old woman, mitochondrial movement was less active, and elongated mitochondria were predominant. No fusion or fission events were observed within the identically sized view and the same timeframe (Fig. 4B, Video 2).

We then employed the Mitometer algorithm to unbiasedly track mitochondrial signals across the 300 live-cell imaging videos (Fig. 4C). Statistical analysis revealed that the frequencies of both fission and fusion events in the advanced-age group were significantly lower than those in the young group (61 vs. 74, P = 0.009; 22 vs. 28, P = 0.01), resulting in no significant difference in the ratio of fission to fusion frequency between the two groups (Fig. 4D). Within the young group, we found no differences in mitochondrial fission frequency, fusion frequency, or the ratio of fission to fusion frequency between the high and low GQER subgroups (Fig. 4D). In contrast, the advanced-age group showed distinct patterns. While there was no significant difference in mitochondrial fusion between the two subgroups, mitochondrial fission frequency in the high GQER subgroup was higher than the low GQER subgroup (68 vs. 57, P = 0.04). This resulted in a significantly higher ratio of fission to fusion frequency in the former compared to the latter (3.4 vs. 2.4, P = 0.01) (Fig. 4D). Correlation analysis showed no significant relationship between GQER and fission or fusion frequency (Fig. 4E and F). However, we observed a positive correlation between GQER and the ratio of fission to fusion frequency (P = 0.01, r = 0.44) (Fig. 4G). These findings suggest that disruption of mitochondrial fission-fusion balance in CCs may lead to decreased early embryonic developmental potential.

Levels of mff and fis1 in CCs positively correlated with GQER

We further investigated differences in mitochondrial dynamic gene expression between advanced-age and young groups, as well as between high and low GQER subgroups, and their correlation with GQER. We selected six representative CC samples from each group for Western blot analysis. Results showed no significant differences in fusion-related protein levels (OPA1, MFN1, and MFN2) between age groups or subgroups (Fig. 5A and B). The fission-related protein DRP1 showed similar levels across groups and subgroups. However, MFF and FIS1 expression levels were significantly higher in the young group compared to the advanced-age group (P = 0.005; P = 0.001) (Fig. 5C and D), and in the high GQER subgroup compared to the low GQER subgroup (MFF: P = 0.03, P = 0.02; FIS1: P = 0.009, P = 0.03) (Fig. 5C and D).

qPCR analysis of 52 CC samples revealed that mRNA expression levels of opa1, mfn1, mfn2, and drp1 were consistent with their protein expression, showing no significant differences between age groups or subgroups (Fig. 5E). Unlike MFF protein expression, mff mRNA levels didn't differ significantly between age groups. However, in the advanced-age group, mff mRNA expression was significantly higher in the high GQER subgroup compared to the low GQER subgroup (P = 0.008) (Fig. 5F). Notably, fis1 mRNA expression was significantly higher in the young group compared to the advanced-age group (P = 0.002). In both age groups, fis1 mRNA expression was significantly higher in the high GQER subgroup compared to the low GQER subgroup (P = 0.04; P = 0.02) (Fig. 5F). Correlation analysis revealed that fusion-related gene expression did not significantly correlate with GQER (Fig. 5G). However, the mRNA expression levels of fission-related genes, mff and fis1, showed a positive correlation with GQER (mff: P = 0.0025, r = 0.41; fis1: P < 0.0001, r = 0.55) (Fig. 5H). These findings suggest that the expression of mitochondrial fission genes in CCs plays a critical role in early embryonic development. Of particular significance is the level of FIS1 expression, which demonstrates the most robust correlation with the GQER among all examined mitochondrial fission-fusion parameters.

Age-related fertility decline accelerates in women over 35 years [27], yet the underlying mechanisms remain unclear. Mitochondria dysfunction is a hallmark of reproductive aging [28, 29], but the dynamic alterations of mitochondria caused by maternal aging in human CCs remain unknown. In this study, we explored the correlations between mitochondrial dynamics of CCs and embryo quality, aiming to provide insights that could extend female reproductive lifespan. A large size of samples was analyzed for robust results, with live-cell imaging techniques employed in order to observe the morphology and dynamics of mitochondria in CCs. Furthermore, we examined the expression of genes related to mitochondrial fusion and fission and found a positive correlation between mff, fis1 expression levels in CCs and embryo developmental potential.

Mitochondrial morphology is closely linked to its functions [18]. Fragmentation typically occurs during nutrient excess, cell apoptosis, mitosis, or increased energy demands [30]. Conversely, elongation serves as a protective response to nutrient depletion, UV irradiation, or cycloheximide exposure [31]. In our study, we observed that mitochondria in CCs from advanced-age women exhibited a consistently elongated morphology compared to young women. This finding aligns with previous observations of hyperfused mitochondria in senescent cells [32–34]. However, Li et al. reported fragmented mitochondria in both senescent human ovarian granulosa cell lines and primary human CCs from women aged ≥ 38 [35]. Transmission electron microscopy results reported abnormal ultrastructure [14], increased volume [15], and elongated shape [36] of mitochondria in aged CCs or granulosa cells. A recent study demonstrated that mitochondrial hyperfusion during senescence helps prevent the minority outer membrane permeabilization and subsequent release of mtDNA [37]. This finding offers a pertinent interpretation for our observations that mitochondrial elongation in aged CCs may represent a protective and compensatory response to the increased membrane permeability and reduced energy production that occur during aging. Furthermore, elongated mitochondria are associated with elevated fatty acid oxidation [38, 39], in line with the upregulated β-oxidation and increased free fatty acids in senescent cells [40].

Mitochondrial dynamics, including fusion and fission, are essential for determining mitochondrial morphology and cellular function [41]. However, the research on mitochondrial dynamics has been constrained by the biased and labor-intensive nature of manual analysis methods. To address these challenges, we employed Mitometer, an open-source tool for automated and robust mitochondrial segmentation and tracking. By this tool, we analyzed fusion and fission events in 30 CC samples through time-lapse imaging and managed to find a reduction in these events within aged CCs. This observation is consistent with previous in silico studies, which reported declining fusion and fission rates with aging [42]. Furthermore, we identified a negative correlation between the ratio of fusion to fission frequency and the GQER, suggesting that maintaining the balance between fusion and fission is necessary for embryo development. Moreover, previous studies showed that mice with both fusion and fission impairments lived longer than those with isolated defects, highlighting that imbalances in fusion-fission may be more detrimental than a general slowdown in mitochondrial dynamics [43, 44].

Mitochondrial fusion and fission are highly regulated processes involving complex molecular machinery. Our study observed no significant age-related alterations in the mRNA expressions of fusion-related genes, aligning with previous findings in human granulosa cells [15]. Notably, both the mRNA and protein expression levels of FIS1 and MFF, which are associated with mitochondrial fission, were lower in the advanced-age group compared to the young group. Furthermore, we observed a positive correlation between mRNA expressions of FIS1, MFF and the GQER. Mitochondrial fission is mediated by the cytoplasmic protein DRP1, which forms multimeric ring complexes at the fission site and facilitates the division of double membranes with the help of actin fibers [19]. DRP1 recruitment relies on two key adaptors on the mitochondrial outer membrane: FIS1 and MFF. FIS1 directs peripheral fission to remove depolarized mitochondria through mitophagy [20]. Conditional knock-out of FIS1 in mice skeletal muscle led to mitochondrial hyperfusion, respiratory chain deficiencies, and impaired mitophagy [45]. Similarly, FIS1 knockdown induced cellular senescence, characterized by mitochondrial elongation and increased ROS level, while its overexpression curbed mitochondrial elongation and reversed senescent phenotypes [46]. On the other hand, MFF governs midzone fission to generate new mitochondria, as part of mitochondrial biogenesis [20]. Mitochondrial biogenesis was observed dramatically reduced in CCs from women with diminished ovarian reserves [47], consistent with our findings of decreased MFF expression in CCs from women with lower quality embryos. Taken together, these findings suggest that reduced expressions of FIS1 and MFF in CCs lead to unopposed fusion and disrupted mitochondrial dynamics, contributing to age-related mitochondrial elongation, compromised mitophagy, and ultimately impaired embryonic development.

The morphology, dynamics, and fission-fusion gene expressions in CC mitochondria are crucial factors impacting early embryo development. Notably, the FIS1 level exhibits the strongest correlation with GQER among these factors. Evaluating the frequency of mitochondrial fission-fusion in cumulus cells might provide a reliable biomarker for prognosticating post-fertilization oocyte progression. Additionally, modulating mitochondrial fission-fusion balance may offer a potential therapy for improving embryo quality in women of advanced age.

CCs

Cumulus cells

GQER

good-quality embryo rate

FIS1

mitochondrial fission protein 1

MFF

mitochondrial fission factor

DRP1

dynamin-related protein 1

MFN1

mitochondrial fusion protein 1

MFN2

mitochondrial fusion protein 2

OPA1

optic atrophy protein 1

ART

assisted reproductive technologies

COCs

Cumulus-oocyte complexes

BMI

body mass index

LH

luteinizing hormone

FSH

follicle-stimulating hormone

E2

estradiol

P4

progesterone

AMH

anti-mullerian hormone

AFC

antral follicle count

PN

pronucleus.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Zhongshan Hospital Fudan University (B2024-185R) and conducted in accordance with the Declaration of Helsinki (as revised in 2013). All the patients provided the written informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Supplementary video 1. Mitochondrial fission and fusion in CCs from a 29-year-old woman.

Live cell imaging of mitochondrial dynamics in CCs labelled with MitoTracker^Red. The video was acquired at 1 frame per 5 seconds for 3 minutes within a 34×34 µm field of view, corresponding to Fig. 4A. Four fission events (indicated by white arrows) and two fusion events (indicated by green arrows) were observed.

Supplementary video 2. Mitochondrial fission and fusion in CCs from a 42-year-old woman.

Live cell imaging of mitochondrial dynamics in CCs labelled with MitoTracker^Red. The video was acquired at 1 frame per 5 seconds for 3 minutes within a 34×34 µm field of view, corresponding to Fig. 4B. No fission or fusion events were observed.

Funding

This study was supported by the National Natural Science Foundation of China (31871506), the Shanghai Science and Technology Commission (19JC1411200), and Shanghai Medical College, Fudan University (yg2023-03).

Author Contribution

Y.S. contributed to the conception and design of the study, as well as data acquisition, analysis, and drafting of the manuscript. S.S. contributed to sample collection, observation, and data analysis. Y.C. contributed to critically revising the manuscript. Z.M. focused on data analysis. Y.L. contributed to the interpretation of the results. H.S. contributed to the conception and design of the experiment. S.L. contributed to the conception and critically reviewed the manuscript.

Acknowledgement

The authors acknowledge Xiaoyang Wang and Haoyuan Tan for their valuable suggestions in image processing, and Ezhou Tang for creating the schematic diagram. The authors extend their gratitude to all members of the embryology team at Zhongshan Hospital for their assistance in collecting CCs.

Data Availability

Data supporting the findings of this article are available upon reasonable request from the corresponding authors.

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No competing interests reported.

SupplementalTable1.docx
SupplementaryFile1.tif
Vedio1.mp4
Supplementary video 1. Mitochondrial fission and fusion in CCs from a 29-year-old woman. Live cell imaging of mitochondrial dynamics in CCs labelled with MitoTracker^Red. The video was acquired at 1 frame per 5 seconds for 3 minutes within a 34×34 μm field of view, corresponding to Fig. 4A. Four fission events (indicated by white arrows) and two fusion events (indicated by green arrows) were observed.
Vedio2.mp4
Supplementary video 2. Mitochondrial fission and fusion in CCs from a 42-year-old woman. Live cell imaging of mitochondrial dynamics in CCs labelled with MitoTrackerRed. The video was acquired at 1 frame per 5 seconds for 3 minutes within a 34×34 μm field of view, corresponding to Fig. 4B. No fission or fusion events were observed.
Vediostill1.tif
Vediostill2.tif

Mitochondrial FIS1 level in cumulus cells correlates with morphological grades of human cleavage-stage embryos

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials and methods

Study participants

Cumulus cells collection and culture

Embryo quality assessment

MitoTracker staining

Live cell imaging

Image processing and analysis

RNA extraction and qRT-PCR analysis

Western blotting

Statistical analysis

Results

Study population, clinical characteristics, and embryology laboratory outcomes

Abnormal mitochondrial morphology in CCs linked to compromised embryo cleavage

Mitochondrial fission-fusion imbalance in CCs associated with reduced GQER

Discussion

Conclusions

Abbreviations

Declarations

Declarations

Competing interests

Supplementary video 1. Mitochondrial fission and fusion in CCs from a 29-year-old woman.

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1