Bersinger Nick A, Eisenhut Markus, Stute Petra, von Wolff Michael
University Women's Hospital, Division of Gynaecological Endocrinology and Reproductive Medicine, University of Berne, Switzerland.
Int J Reprod Med. 2021 Jan 27;2021:2906164. doi: 10.1155/2021/2906164. eCollection 2021.
The follicular fluid (FF) plays an essential role in the physiology of the follicle and the oocyte. Gonadotropin stimulation affects the FF steroid hormone and anti-Mullerian hormone (AMH) concentrations, which has been suggested to be the reason for lower oocyte competence in conventional gonadotropin stimulated fertilisation (cIVF) compared to natural cycle IVF (NC-IVF). To analyse the effect of gonadotropin stimulation on a broad spectrum of signalling proteins, we ran proteomic antibody arrays on FF of women undergoing both treatments NC-IVF and cIVF.
Twenty women underwent one NC-IVF and one cIVF treatment cycle. Follicular fluids of the first aspirated follicle were compared between the two groups using a protein microarray which included antibodies against 224 proteins related to cell signalling and reference proteins. Each of the 40 albumin-stripped, matched-pair samples was labelled in the reverse-dye (Cy3/Cy5) procedure before undergoing array hybridisation. Signal analysis was performed using normalisation algorithms in dedicated software. Five proteins yielding a value of < 0.05 in the array experiment (Cystatin A, Caspase-3, GAD65/67, ERK-1, and ERK-2) were then submitted to quantitative determination by ELISA in the same follicular fluids.
Array analysis yielded only a small number of differentially expressed signalling markers by unadjusted values. Adjustment as a consequence of multiple determinations resulted in the absence of any significant differential marker expression on the array. Five unadjusted differentially expressed proteins were quantified immunometrically with antibodies from different sources. Follicular fluid concentrations of Cystatin A and MAP kinase ERK-1 concentrations were significantly higher in the cIVF than in the NC-IVF follicles, while GAD-2 (GAD65/67) did not differ. The assays for Caspase-3 and MAP kinase ERK-2 did not have the required sensitivities.
In contrast to FF steroid hormones and AMH, FF concentrations of signalling proteins are not or only marginally altered by gonadotropin stimulation.
卵泡液（FF）在卵泡和卵母细胞的生理过程中起着至关重要的作用。促性腺激素刺激会影响卵泡液中甾体激素和抗苗勒管激素（AMH）的浓度，这被认为是与自然周期体外受精（NC-IVF）相比，传统促性腺激素刺激体外受精（cIVF）中卵母细胞能力较低的原因。为了分析促性腺激素刺激对多种信号蛋白的影响，我们对接受NC-IVF和cIVF两种治疗的女性的卵泡液进行了蛋白质组抗体芯片检测。
20名女性接受了一个NC-IVF和一个cIVF治疗周期。使用包含针对224种与细胞信号相关的蛋白质和参考蛋白质的抗体的蛋白质微阵列，比较两组中首次抽吸卵泡的卵泡液。在进行芯片杂交之前，将40个去除白蛋白的匹配样本中的每一个在反向染料（Cy3/Cy5）程序中进行标记。使用专用软件中的归一化算法进行信号分析。然后，对在芯片实验中值<0.05的5种蛋白质（胱抑素A、半胱天冬酶-3、谷氨酸脱羧酶65/67、细胞外信号调节激酶-1和细胞外信号调节激酶-2）在相同的卵泡液中进行酶联免疫吸附测定（ELISA）定量测定。
芯片分析未校正值仅产生少量差异表达的信号标记物。由于多次测定的校正导致芯片上没有任何显著的差异标记物表达。使用来自不同来源的抗体对5种未校正的差异表达蛋白质进行免疫测定定量。cIVF卵泡中胱抑素A和丝裂原活化蛋白激酶细胞外信号调节激酶-1的卵泡液浓度显著高于NC-IVF卵泡，而谷氨酸脱羧酶-2（谷氨酸脱羧酶65/67）无差异。半胱天冬酶-3和丝裂原活化蛋白激酶细胞外信号调节激酶-2的检测没有所需的灵敏度。
与卵泡液甾体激素和AMH不同，促性腺激素刺激对卵泡液中信号蛋白的浓度没有影响或仅有轻微改变。
