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Mitochondrial metabolic substrate utilization in granulosa cells reflects body mass index and total follicle stimulating hormone dosage in in vitro fertilization patients

  • Assisted Reproduction Technologies
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Abstract

Purpose

To utilize a novel mitochondrial function assay with pooled granulosa cells to determine whether mitochondrial function would differ by patient demographics and embryo development.

Methods

This was a prospective pilot study in a hospital-based assisted reproductive program and public university. Mitochondrial metabolic substrate utilization was assessed in pooled granulosa cells from 40 women undergoing in vitro fertilization during 2018 and 2019.

Results

Assessment of mitochondrial substrate metabolism in pooled granulosa cells revealed higher citric acid, L-malic acid, and octanoyl-L-carnitine utilization with higher body mass index (BMI). Utilization of citric acid, cis-aconitic acid, D-alpha-keto-glutaric acid, L-glutamine, and alanine plus glycine was significantly lower as total dosage of FSH administered increased. Utilization of glycogen was significantly higher in patients with a higher percentage of fertilized oocytes. D-alpha-keto-glutaric acid utilization was significantly lower in patients with a higher percentage of good 8-cell embryos. L-glutamine utilization was significantly lower, with a higher percentage of blastocyst formation. Mitochondrial metabolic scores (MMS), which reflect overall mitochondrial activity of the granulosa pool, were significantly higher in patients with higher BMI and with greater numbers of mature oocytes retrieved. MMS in granulosa decreased as total FSH dose administered increased.

Conclusions

Granulosa cell utilization of substrates feeding into the citric acid cycle changed with total FSH dosage and BMI. Fertilization rate, 8-cell embryo quality, and blastocyst formation also associated with different energy substrate usage. Mitochondrial substrate utilization by granulosa cells from individual follicles could be further developed into a useful diagnostic tool.

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Funding

Supported by an ASPIRE-I grant from the University of South Carolina.

Author information

Authors and Affiliations

  1. Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC, USA

    Richard J. Kordus & Holly A. LaVoie

  2. Department of Obstetrics and Gynecology, Fertility Center of the Carolinas, Prisma Health – Upstate, Greenville, SC, USA

    Richard J. Kordus & Henry E. Malter

  3. Department of Epidemiology and Biostatistics, University of South Carolina, Columbia, SC, USA

    Akhtar Hossain

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  1. Richard J. Kordus
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Correspondence to Holly A. LaVoie.

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Conflict of interest

The authors declare no conflict of interest related to this study.

Ethical approval

“All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and in its later amendments or comparable ethical standards.” This study was approved by the Greenville Health System (IRB number: Pro00072006) and the University of South Carolina Institution Review Board (IRB registration number: 00000240).

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Informed consent was obtained from all individual participants included in the study.

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Electronic supplementary material

Supplementary data are available at the Journal of Assisted Reproduction and Genetics online.

ESM 1

Plot of granulosa cell number per well versus the difference in absorbance between the positive and negative controls for the Mitoplate S-1 assays confirm the linear relationship. Data include n = 45 patients including those used in optimization experiments. Patients showing no absorbance difference between the controls at approximately 6500 cells or less were not included. (PDF 230 kb)

ESM 2

Substrates that demonstrated no significant mitochondrial utilization differences based on body mass index (BMI). To determine the differences in mitochondrial utilization, substrates were compared using a multiple linear regression model using Box-Cox transformation between BMI groups. BMI was divided into 2 groups with the higher group being patients with a BMI of ≥ to 26 kg/m2 (n = 20) and the lower group being patients with a BMI of <26 kg/m2 (n = 20). Mitochondrial utilization of cis-aconitic acid (P < 0.10), D,L-beta-hydroxy-butyric acid (P < 0.10), and pyruvic acid with L-malic acid 100 μM (P < 0.10) trended to be higher in pooled GCs of patients with higher BMI, after adjusting for the other factors in the model. No other substrates showed any mitochondrial utilization differences. Following the Box-Cox transformation of the data and observing the Q-Q plots, the most outlying observations have been removed to ensure the normality of the response variables. Data are presented as the median (line inside box), first and third quartile (bottom and top of the box), and highest and lowest data points (top and bottom of whiskers). (PDF 8162 kb)

ESM 3

Substrates that demonstrated no significant mitochondrial utilization differences based number (A) and percentage (B) of mature oocytes retrieved. To determine the differences in mitochondrial utilization, substrates were compared using a multiple linear regression model using Box-Cox transformation. No substrates in pooled GCs of patients demonstrated any significant mitochondrial utilization differences after adjusting for the other factors in the model. Following the Box-Cox transformation of the data and observing the Q-Q plots, the most outlying observations have been removed to ensure the normality of the response variables. Data are presented as a scatter plot of the individual patient values lambda transformed for normalization showing the trend line and the 95% confidence interval (PDF 8525 kb)

ESM 4

Substrates that demonstrated no significant mitochondrial utilization differences based on total FSH dose administered. To determine the differences in mitochondrial utilization, substrates were compared using a multiple linear regression model using Box-Cox transformation. Mitochondrial utilization of D,L-isocitric acid (P < 0.10), D,L-beta-hydroxy-butyric acid (P < 0.10), L-glutamic acid (P < 0.10), acetyl-L-carnitine (P < 0.10), pyruvic acid with L-malic acid 100 μM (P < 0.10) trended to be lower in pooled GCs of patients with higher administered FSH dosages, after adjusting for the other factors in the model. No other substrates showed any mitochondrial utilization differences. Following the Box-Cox transformation of the data and observing the Q-Q plots, the most outlying observations have been removed to ensure the normality of the response variables. Data are presented as a scatter plot of the individual patient values lambda transformed for normalization showing the trend line and the 95% confidence interval (PDF 6449 kb)

ESM 5

Substrates that demonstrated no significant mitochondrial utilization differences based fertilization percentage. To determine the differences in mitochondrial utilization, substrates were compared using a multiple linear regression model using Box-Cox transformation. The two groups were delineated by higher fertilization percentage ≥ 80% (n = 22), and lower fertilization percentage < 80% (n = 18). Following the Box-Cox transformation of the data and observing the Q-Q plots, the most outlying observations have been removed to ensure the normality of the response variables. Data are presented as the median (line inside box), first and third quartile (bottom and top of the box), and highest and lowest data points (top and bottom of whiskers) (PDF 7626 kb)

ESM 6

Substrates that demonstrated no significant mitochondrial utilization differences based on good 8-cell embryo development. To determine the differences in mitochondrial utilization, substrates were compared using a multiple linear regression model using Box-Cox transformation. The two groups were delineated by higher good 8-cell development >55% (n = 22) and lower good 8-cell development ≤55% (n = 18). Mitochondrial utilization of L-serine (P < 0.10) trended to be lower in GCs of patients with a higher percentage of good quality 8-cell embryos. In a backward selected best fit model using only the percentage of good 8-cell embryos and percentage of blastocysts, L-glutamine (P < 0.10) trended to be lower in pooled GCs from patients with a higher percentage of good quality 8-cell embryos. No other substrates showed significant differences. Following the Box-Cox transformation of the data and observing the Q-Q plots, the most outlying observations have been removed to ensure the normality of the response variables. Data are presented as the median (line inside box), first and third quartile (bottom and top of the box), and highest and lowest data points (top and bottom of whiskers) (PDF 7199 kb)

ESM 7

Substrates that demonstrated no significant mitochondrial utilization differences based on percent blastocyst formation. To determine the differences in mitochondrial utilization, substrates were compared using a multiple linear regression model using Box-Cox transformation. The two groups were delineated by higher percentage of blastocyst formation >55% (n = 17) and lower percentage of blastocyst formation ≤55% (n = 22). Granulosa mitochondrial utilization of D-Gluconate-6-phosphate (P < 0.10) and D,L-isocitric acid (P < 0.10) trended to be lower in patients with a higher percentage of blastocyst formation, after adjusting for the other factors in the model. No other substrates demonstrated significant differences. Following the Box-Cox transformation of the data and observing the Q-Q plots, the most outlying observations have been removed to ensure the normality of the response variables. Data are presented as the median (line inside box), first and third quartile (bottom and top of the box), and highest and lowest data points (top and bottom of whiskers) (PDF 7111 kb)

ESM 8

Pooled granulosa cell mitochondrial DNA (mtDNA) content had no significant association with patient demographics nor oocyte outcomes. Correlation analysis showed mtDNA was similar between all groups. For categorical groups, data are presented as the median (line inside box), first and third quartile (bottom and top of the box), and highest and lowest data points (top and bottom of whiskers). For continuous variables, data are presented as a scatter plot of the individual patient values lambda transformed for normalization showing the trend line and the 95% confidence interval. 8-cells = 8-cell embryos, AFC = antral follicle count, AMH = anti-Mullerian hormone, ANT = antagonist stimulation, BMI = body mass index, FSH = follicle stimulating hormone, HMG = human menopausal gonadotropin, LL = long luteal stimulation, STIM = stimulation type (PDF 3804 kb)

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Kordus, R.J., Hossain, A., Malter, H.E. et al. Mitochondrial metabolic substrate utilization in granulosa cells reflects body mass index and total follicle stimulating hormone dosage in in vitro fertilization patients. J Assist Reprod Genet 37, 2743–2756 (2020). https://doi.org/10.1007/s10815-020-01946-9

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