Detti Laura, Fletcher Nicole M, Saed Ghassan M, Peregrin-Alvarez Irene, Uhlmann Rebecca A
1 University of Tennessee Health Science Center, Department of Obstetrics and Gynecology, Memphis, TN, USA.
2 The C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, MI, USA.
Reprod Sci. 2018 Aug;25(8):1218-1223. doi: 10.1177/1933719117737850. Epub 2017 Nov 15.
To test whether recombinant anti-Müllerian hormone (AMH) can inhibit ovarian cortex function by modulating the expression of other hormone receptors.
Pilot experimental study with ovarian cortex obtained from 5 patients. Immediately after explant, the ovarian cortex specimens were divided into 5 equal fragments. One fragment was flash-frozen (uncultured) and 4 were incubated for 48 hours at 37°C in a pH-adjusted gamete buffer medium with increasing AMH concentrations of 0, 5, 25, and 50 ng/mL. After incubation, all specimens were rinsed and flash-frozen for polymerase chain reaction (PCR) executed in triplicates. We utilized real-time reverse transcription-polymerase chain reaction (RT-PCR) to determine messenger RNA (mRNA) levels of AMH and its receptor Anti-Müllerian Hormone-Receptor 2 (AMH-R2), follicle stimulating hormone receptor (FSH-R), luteinizing hormone receptor (LH-R), inhibin B, and insulin-like growth factor 1 receptor 1 (IGF1-R1) in ovarian cortex tissue. In addition, we performed Ki-67 immunostaining to evaluate cell proliferation in the treatment groups.
Absence of recombinant human AMH (rAMH) caused upregulation of all markers. Exposure to increasing rAMH concentrations caused tissue AMH expression downregulation ( P = .024), while AMH-R2 ( P = .005), FSH-R ( P = .009), LH-R ( P = .003), and inhibin B ( P = .001) mRNA expression followed a bell-shaped response with an increased expression at low dose, followed by a decreased expression at higher doses. Expression of IGF1-R1 was independent ( P = .039) of rAMH exposure. The Ki-67 immunostaining showed an increased cell proliferation in the media control compared to the uncultured and the tissue cultured with rAMH.
Culture with increasing rAMH concentrations caused downregulation of its own, as well as other hormone receptors, and a decreased ovarian cortex cell proliferation. These results help understanding the inhibitory effects of AMH on follicular development.
检测重组抗苗勒管激素（AMH）是否可通过调节其他激素受体的表达来抑制卵巢皮质功能。
对取自5例患者的卵巢皮质进行初步实验研究。取出后，立即将卵巢皮质标本分成5个相等的碎片。其中一个碎片速冻保存（未培养），另外4个碎片在37°C下于pH值调整后的配子缓冲培养基中培养48小时，培养基中AMH浓度分别为0、5、25和50 ng/mL。培养后，所有标本冲洗后速冻，进行三次重复的聚合酶链反应（PCR）。我们利用实时逆转录聚合酶链反应（RT-PCR）来测定卵巢皮质组织中AMH及其受体抗苗勒管激素受体2（AMH-R2）、促卵泡激素受体（FSH-R）、促黄体生成素受体（LH-R）、抑制素B和胰岛素样生长因子1受体1（IGF1-R1）的信使核糖核酸（mRNA）水平。此外，我们进行Ki-67免疫染色以评估各治疗组中的细胞增殖情况。
无重组人AMH（rAMH）时所有标志物上调。暴露于浓度递增的rAMH中导致组织AMH表达下调（P = 0.024），而AMH-R2（P = 0.005）、FSH-R（P = 0.009）、LH-R（P = 0.003）和抑制素B（P = 0.001）的mRNA表达呈钟形反应，低剂量时表达增加，高剂量时表达降低。IGF1-R1的表达与rAMH暴露无关（P = 0.039）。Ki-67免疫染色显示，与未培养及用rAMH培养的组织相比，培养基对照中的细胞增殖增加。
用浓度递增的rAMH培养导致其自身以及其他激素受体下调，卵巢皮质细胞增殖减少。这些结果有助于理解AMH对卵泡发育的抑制作用。
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