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. 2016 Jun;31(6):1315-26.
doi: 10.1093/humrep/dew048. Epub 2016 Apr 6.

Diet-induced Obesity Impairs Endometrial Stromal Cell Decidualization: A Potential Role for Impaired Autophagy

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Free PMC article

Diet-induced Obesity Impairs Endometrial Stromal Cell Decidualization: A Potential Role for Impaired Autophagy

Julie S Rhee et al. Hum Reprod. .
Free PMC article

Abstract

Study question: What effect does diet-induced obesity have on endometrial stromal cell (ESC) decidualization?

Summary answer: Diet-induced obesity impairs ESC decidualization.

What is known already: Decidualization is important for successful implantation and subsequent health of the pregnancy. Compared with normal-weight women, obese women have lower pregnancy rates (both spontaneous and by assisted reproductive technology), higher rates of early pregnancy loss and poorer oocyte quality.

Study design, size, duration: Beginning at 6 weeks of age, female C57Bl/6J mice were fed either a high-fat/high-sugar diet (HF/HS; 58% Fat Energy/Sucrose) or a diet of standard mouse chow (CON; 13% Fat) for 12 weeks. At this point, metabolic parameters were measured. Some of the mice (n = 9 HF/HS and 9 CON) were mated with reproductively competent males, and implantation sites were assessed. Other mice (n = 11 HF/HS and 10 CON) were mated with vasectomized males, and artificial decidualization was induced. For in vitro human studies of primary ESCs, endometrial tissue was obtained via biopsy from normo-ovulatory patients without history of infertility (obese = BMI > 30 kg/m(2), n = 11 and lean = BMI < 25 kg/m(2), n = 7) and from patients consented for hysterectomies for a benign indication (n = 4). In vitro studies were also performed with immortalized human ESCs. ESCs were decidualized in culture for nine 9 days in the presence or absence of palmitic acid (PA), and the degree of decidualization was assessed by measuring expression of decidualization markers.

Participants/materials, setting, methods: The sizes of implantation sites and fetuses were analyzed in mice mated with reproductively competent males. In mice mated with vasectomized males, decidualization was induced, and uterine tissues were analyzed via hematoxylin and eosin staining, quantitative RT-PCR (RT-qPCR), and western blots. Human ESCs were cultured in vitro and induced to decidualize by treatment with cAMP and medroxyprogesterone. The level of expression of decidualization markers was assessed by RT-qPCR (mRNA) and western blotting (protein). ATP content of ESCs was measured, and levels of autophagy were assessed by western blotting of the autophagy regulators acetyl coa carboxylase (ACC) and ULK1 (Ser 317). Autophagic flux was measured by western blot of the marker LC3b-II.

Main results and the role of chance: Mice exposed to an HF/HS diet became obese and metabolically impaired. HF/HS-exposed mice mated to reproductively competent males had smaller implantation sites in early pregnancy (P <0.001) and larger fetuses at term (P <0.05) than CON-exposed mice. In the artificial decidualization experiments, mice exposed to the HF/HS diet developed 50% smaller deciduomas than mice exposed to CON diet (P< 0.001). Human ESCs cultured in the presence of PA had markedly decreased mRNA expression of the decidualization markers, decidual prolactin (PRL) (P< 0.0001) and insulin-like growth factor binding protein 1 (IGFBP1) (P< 0.0001). Expression of PRL and IGFBP1 by mRNA were also significantly lower in early follicular phase ESCs of obese women than in those of normal-weight women (P< 0.05). Protein expression of phosphorylated ACC and phosphorylated ULK1, both activated forms, were lower in deciduomas of HF/HS mice than in those of control mice (P < 0.01). In immortalized human ESCs, LC3b-II levels were higher in decidualized cells than in controls, indicating increased autophagy. PA treatment abrogated this increase.

Limitations, reasons for caution: Many aspects of obesity and metabolic impairment could contribute to the decidualization defects observed in the HF/HS-exposed mice. Although our findings suggest that both autophagy and decidualization are impaired by exposure to PA, the underlying mechanisms should be elucidated. Finally, our human patient sample size was small.

Wider implications of the findings: Although many factors contribute to poor reproductive outcome and early pregnancy loss in obese women, our study suggests the importance of decidualization defects. Such defects may contribute to compromised endometrial receptivity and poor implantation. If defects in autophagy contribute to impaired decidualization, therapeutics could be developed to improve this process and thus improve implantation and pregnancy outcomes in obese women.

Study funding/competing interests: Grants include NIH 5T32HD040135-12 (J.S.R.), R01 HD065435 (K.H.M.), NIH T32 HD049305 (J.L.S.) and ACOG Research Grant (M.B.S.). The authors report no conflicts of interest.

Keywords: autophagy; decidualization; implantation; obesity; reproduction.

Figures

Figure 1
Figure 1
High-fat/high-sucrose feeding leads to obesity and altered glucose metabolism in mice. (a) Body weight, (b) fat and lean body mass, (c) glucose tolerance test, (d) serum insulin levels and (e) serum lipids [cholesterol, non-esterified fatty acids (NEFA) and triglycerides (TG)] measured after 12 weeks of control (CON) or high-fat/high-sucrose (HF/HS) diet. Values are expressed as mean ± SE. *P < 0.01, as analyzed by Student’s t-tests. n = 27 control fed mice and n = 25 HF/HS-fed mice.
Figure 2
Figure 2
Diet-induced obesity in mice leads to altered implantation, size and shape of sites in early pregnancy and fetal macrosomia at term. (a) Implantation site number at dpc 5.5 (n = 6 mice in each group); (b) at dpc 8.5 (n = 14 mice I each group); and (c) at dpc 18.5 (n = 5 in each group). (d) Representative images of implantation sites at dpc 8.5 from CON- and HF/HS-exposed mice. (e) Diameter and weight at dpc 8.5 (n = 14 mice in each group). (f) Representative images of term (dpc 18.5) CON and HF/HS fetuses (n = 5 each group). (g) Fetal and placental weights on dpc 18.5 (n = 5 mice in each group). Values are expressed as mean ± SE. *P < 0.05, ***P < 0.0001 as analyzed by the Mann–Whitney U-test.
Figure 3
Figure 3
HF/HS diet impairs artificial ESC decidualization in mice. (a) Representative images of deciduomas formed in pseudo-pregnant CON- and HF/HS-exposed mice. (b) Weights and (c) representative cross-sections of deciduomas. (d) mRNA expression level of Prp in deciduomas (n = 10 Con, n = 11 HF/HS). Values are expressed as mean ± SE. *P < 0.05, as analyzed by the Mann–Whitney U-test.
Figure 4
Figure 4
Autophagy is up-regulated during murine decidualization. (a) Representative western blots show relative protein levels of acetyl-CoA carboxylase (ACC), phosphorylated-ACC, phosphorylated-Unc-like kinase 1 (ULK1), ULK1 and loading control GAPDH in unstimulated (US) and stimulated (S) uterine horns from CON- and HF/HS-exposed mice. (b) Quantification of band intensities (n = 6–8 mice per group). Values are expressed as mean ± SE. *P < 0.01 between the Con and HF/HS groups, $P < 0.05 between the US and S groups. The Mann–Whitney U-test was used to analyze the data.
Figure 5
Figure 5
PA impairs human ESC decidualization in vitro. Expression levels of IGFBP1 and decidual prolactin (PRL) mRNA in (a) immortalized and (b) primary human ESCs in the presence or absence of PA. (c) Expression of IGFBP1 and PRL mRNA in decidualized endometrial cells from obese (BMI > 30 kg/m2) and lean (BMI < 25 kg/m2) women (n = 11 obese, n = 7 lean). Values are expressed as mean fold change over undecidualized ± SE. *P < 0.001 as analyzed by the Mann–Whitney U-test.
Figure 6
Figure 6
Autophagy is up-regulated during human ESC decidualization. (a) Western blot of LC3b-II and GAPDH (loading control) in control (Con) and decidualized (Dec) human ESCs in the presence or absence of PA and in the presence or absence of Bafilomycin A1 (BafA1). (b) Quantification of LC3b-II level relative to GAPDH in BafA1-treated cells (n = 3–7 treatment wells per condition). Values are expressed as mean ± SE. *P<0.001, as analyzed by analysis of variance with the Bonferroni correction for multiple comparisons. This experiment was performed in triplicate.
Figure 7
Figure 7
ATP levels decrease during human ESC decidualization. ATP concentration in control (Con) and decidualized (Dec) human ESCs in the presence or absence of PA (n = 3 treatment wells per condition). Values are expressed as mean ± SE. *P< 0.001 as analyzed by Student’s t-tests.

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