Supplementary Material for: ERRγ1 and Aromatase Expression in Human Placental Tissues from Term Deliveries of Large for Gestational Age (LGA) Newborns

Introduction: Being born either large (LGA) or small for gestational age (SGA) has been associated with an increased risk of developing metabolic syndrome in adulthood. However, the mechanism underlying this early programming remained unclear.
Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor with a high expression in human placenta, particularly ERRγ1. ERRγ has been proposed to play a central role in controlling genes involved in energy metabolism. In placenta, ERRγ1 acts as an oxygen-responsive transcription factor regulating aromatase (Aro) expression during trophoblast differentiation. Aromatase is an enzyme that catalyzes the synthesis of estrogens from androgens and is located in the syncytiotrophoblast. An adequate estrogen-androgen balance is required for normal pregnancy progression. Our aim was to analyze ERRγ1 and Aro mRNA in human placenta from term LGA newborns. We propose that ERRγ1 and CYP19A1 expressions in human placenta from LGA newborns are impaired, which would modify fetal programming of LGA newborns, since an imbalance in intrauterine estrogen-androgen ratio would be occurred
Methods: Total RNA was obtained from placental tissues of LGA (GA: 39-41 weeks, n=8) and adequate for gestational age (AGA; 39-40 weeks, n=10) newborns. ERRγ1 and Aro mRNA variants were analyzed by RT2-PCR. Primers for Aro analysis were specific for Total aromatase (TotalAro) binding in exons 2-3 and for Active aromatase (ActAro) in exons 9-10. Aro protein was analyzed by Western-blot.
Results: ERRγ1 mRNA was significantly higher in LGA compare to AGA. TotalAro mRNA was significantly lower in LGA in comparison with AGA control. Similar results with Aro protein. In contrast ActAro/TotalAro ratio was higher in LGA compared to the AGA control.
Conclusions: High expression of ERRγ1 as well as ActAro/TotalAro ratio in LGA suggests that ERRγ1 is involved in ActAro variant expression and hence disrupted estrogen-androgen balance in the intrauterine environment.
We propose that dysregulation of ERRγ1 in placenta might modify the estrogen-androgen balance in the intrauterine environment in LGA newborns, possibly representing one of the key factors in the regulation of fetal programming.

引言：胎儿出生时为大于胎龄儿（large for gestational age, LGA）或小于胎龄儿（small for gestational age, SGA），均与成年后罹患代谢综合征的风险升高相关。然而，这一早期发育编程背后的分子机制仍未明确。
雌激素相关受体γ（estrogen-related receptor gamma, ERRγ）是一类在人胎盘中高表达的孤儿核受体，其中ERRγ1亚型的表达尤为显著。已有研究证实，ERRγ在调控能量代谢相关基因的过程中发挥核心作用。在胎盘中，ERRγ1作为氧应答转录因子，可在滋养层细胞分化阶段调控芳香化酶（aromatase, Aro）的表达。芳香化酶是一种可催化雄激素向雌激素合成的酶，定位于合体滋养层细胞。正常妊娠的顺利进行有赖于雌激素与雄激素的适当平衡。本研究旨在分析足月LGA新生儿胎盘组织中ERRγ1与Aro mRNA的表达情况。我们推测，LGA新生儿胎盘中ERRγ1及CYP19A1的表达存在异常，这将引发宫内雌激素-雄激素比例失衡，进而改变LGA新生儿的胎儿发育编程。

方法：本研究从LGA新生儿（胎龄39~41周，n=8）及适于胎龄儿（adequate for gestational age, AGA；胎龄39~40周，n=10）的胎盘组织中提取总RNA。通过RT2-PCR对ERRγ1与Aro mRNA变体进行检测：用于Aro分析的引物分别靶向外显子2~3区域的总芳香化酶（TotalAro），以及外显子9~10区域的活性芳香化酶（ActAro）。同时采用Western-blot检测Aro蛋白的表达水平。

结果：与AGA对照组相比，LGA组胎盘中ERRγ1 mRNA的表达水平显著升高。总芳香化酶（TotalAro）mRNA的表达水平则显著低于AGA对照组，Aro蛋白的检测结果与此一致。与之相反，LGA组的活性芳香化酶与总芳香化酶的比值（ActAro/TotalAro）显著高于AGA对照组。

结论：LGA组胎盘中ERRγ1的高表达以及ActAro/TotalAro比值的升高，提示ERRγ1参与了ActAro亚型的表达调控，进而破坏了宫内环境中的雌激素-雄激素平衡。
我们推测，胎盘中ERRγ1的表达失调可能改变LGA新生儿宫内环境的雌激素-雄激素平衡，这或许是调控胎儿发育编程的关键因素之一。