泌乳素在不明原因复发性流产和正常妊娠早期中的差异性表达

doi:10.3969/j.issn.1674-8115.2021.04.012

摘要/Abstract

摘要：

目的·探讨不明原因复发性流产（unexplained recurrent pregnancy loss，URPL）和正常妊娠早期，泌乳素（prolactin，PRL）在外周血和蜕膜基质细胞（decidual stromal cells，DSCs）中的表达差异。方法·纳入2018年3月—2019年3月于上海交通大学医学院附属仁济医院妇产科就诊的80例URPL患者、70例人工流产妇女以及190例正常妊娠妇女。采用酶联免疫吸附方法检测外周血PRL水平。采用实时定量聚合酶链式反应（quantitative real time PCR，RT-qPCR）、蛋白质免疫印迹（Western blotting，WB）及免疫组化染色（immunohistochemistry，IHC）方法检测DSCs中PRL的mRNA和蛋白表达水平。2组之间比较采用独立样本t检验，多组组间比较采用单因素方差分析。结果·排除21例胚胎染色体异常，UPRL组最终纳入59例。人工流产组纳入70例。190例正常妊娠妇女中177例完成随访，其中157例活产，被归于正常妊娠活产组，其余20例发生胚胎停育或自然流产，其中流产发生于孕10周以前的19例被归入正常妊娠流产组。正常妊娠活产组和人工流产组妇女的外周血PRL水平类似，相同孕周间差异无统计学意义，且都随着孕周增长显著升高（正常妊娠活产组：孕5~5⁺⁶周vs孕6~7⁺⁶周，P=0.002；孕6~7⁺⁶周vs孕8~9⁺⁶周，P= 0.012。人工流产组：孕5~5⁺⁶周vs孕6~7⁺⁶周，P = 0.015，孕6~7⁺⁶周vs孕8~9⁺⁶周，P= 0.023）；URPL患者的外周血PRL水平随着孕周增长无显著变化，并且从孕6周起显著低于相同孕周的正常妊娠活产妇女和人工流产妇女（孕6~7⁺⁶周：P = 0.018，0.024；孕8~9⁺⁶周：P = 0.015，0.003）；正常妊娠流产组的外周血PRL水平也与相同孕周的URPL组患者类似。人工流产组妇女DSCs的PRL表达在mRNA和蛋白水平上都随着孕周增长显著升高（孕5~5⁺⁶周vs 孕6~7⁺⁶周：RT-qPCR，P = 0.020 ；WB，P = 0.010 ；IHC，P = 0.030。孕6~7⁺⁶周vs 孕8~9⁺⁶周：RT-qPCR，P = 0.011；WB，P = 0.034；IHC，P = 0.012）；URPL组DSCs的PRL表达水平增长迟滞：孕6~7⁺⁶周及孕8~9⁺⁶周的URPL妇女DSCs的PRL表达水平显著低于相同孕周的人工流产妇女（孕6~7⁺⁶周：RT-qPCR，P = 0.012；WB，P = 0.014；IHC，P = 0.028；孕8~9⁺⁶周：RT-qPCR，P = 0.011；WB，P = 0.030；IHC，P = 0.026）。结论·UPRL患者DSCs的PRL表达缺陷，DSCs蜕膜化不足可能与URPL的发生有关；孕6周以后外周血PRL低水平可能预示胚胎发育不良。

关键词: 泌乳素, 不明原因复发性流产, 蜕膜基质细胞, 蜕膜化

Abstract:

Objective·To investigate the difference of prolactin (PRL) expression in peripheral blood and decidual stromal cells (DSCs) between unexplained recurrent pregnancy loss (URPL) and normal early pregnancy.

Methods· Eighty cases of URPL patients in our hospital from March 2018 to March 2019 were recruited. Seventy women with induced abortion and 190 normal pregnancy women were selected as controls. Fasting peripheral blood and decidua tissues were collected. The level of PRL in peripheral blood was detected by enzyme-linked immunosorbent assay. Quantitative real time PCR (RT-qPCR), Western bloting (WB) and immunohistochemistry (IHC) were used to detect the PRL expression of DSCs. Independent sample t test was used to compare the data of the two different groups, and one-way analysis of variance test was used to compare the data of the groups.

Results· In the URPL group, 21 cases were excluded for chromosomal abnormality, and 59 cases were finally included. There were 70 cases in the induced abortion group. One hundred and seventy-seven cases in the normal pregnancy group completed follow-up, of which 157 cases were classified as final live birth in the normal pregnancy group, and the rest 20 cases subsequently had pregnancy loss, of which 19 miscarriages occurred in the first 10 weeks of gestation, classified as pregnancy loss in the normal pregnancy group. The PRL levels of peripheral blood in the final live birth in the normal pregnancy group and induced abortion group were similar, with no significant difference between the two groups in the same gestation, and they all increased significantly with advancing gestation (normal pregnancy with final live birth group: 5?5⁺⁶ week vs 6?7⁺⁶ week, P = 0.002; 6?7⁺⁶ week vs 8?9⁺⁶ week,P = 0.012;Induced abortion group: 5?5⁺⁶ week vs 6?7⁺⁶ week, P = 0.015; 6?7⁺⁶ week vs 8?9⁺⁶ week, P = 0.023). The levels of PRL in the peripheral blood of patients with URPL had no significant change with advancing gestation, and they were significantly lower than those of women with final live birth in the normal pregnancy group and induced abortion women in the same gestation from the 6th week (6?7⁺⁶week: P = 0.012, 0.024; 8?9⁺⁶ week: P = 0.015, 0.003). In addition, the levels of PRL in the peripheral blood of the women in the normal pregnancy group who later suffered miscarriage were similar to those in the URPL group of the same gestation. The PRL expression of DSCs in the induced abortion group increased significantly in both RNA and protein level with advancing gestation (5?5⁺⁶ week vs 6?7⁺⁶ week: RT-qPCR, P = 0.020; WB, P = 0.010; IHC,P = 0.030. 6?7⁺⁶ week vs 8?9⁺⁶ week: RT-qPCR, P = 0.011; WB, P = 0.034; IHC, P = 0.012). However, the increase of PRL expression of DSCs in the URPL group was delayed: the PRL expression of DSCs in women with 6?7⁺⁶week and 8?9⁺⁶ week of gestation was significantly lower than that in women accepting induced abortion of the same gestation (6?7⁺⁶ week: RT-qPCR, P = 0.012; WB, P = 0.014; IHC,P = 0.028. 8?9⁺⁶ week: RT-qPCR, P = 0.011; WB, P = 0.030;IHC, P = 0.026).

Conclusion· PRL expression of the decidua is defective. The PRL expression of DSCs in URPL patients is deficient, and the lack of decidualization of DSCs may be related to the occurrence of URPL. The low level of PRL in peripheral blood after 6 weeks of gestation may indicate embryonic dysplasia.

Key words: prolactin, unexplained recurrent pregnancy loss, decidual stromal cell, decidualization

中图分类号:

R714.21

陈超, 李聪聪, 郭枫, 王巧红, 赵爱民. 泌乳素在不明原因复发性流产和正常妊娠早期中的差异性表达[J]. 上海交通大学学报(医学版), 2021, 41(4): 489-496.

Chao CHEN, Cong-cong LI, Feng GUO, Qiao-hong WANG, Ai-min ZHAO. Different expression of prolactin in unexplained recurrent pregnancy loss and normal early pregnancy[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 489-496.

图/表 8

图1 原代蜕膜基质细胞的分离提取

Fig 1 Isolation of primary decidual stromal cells

表1 RT-qPCR引物系列（5′→3′）

Tab 1 Primer sequences for RT-qPCR（5′→3′）

Gene	Specie	Primer sequence
PRL	Human	Forward	GCAGATGGCTGATGAAGAGTCTCG
PRL	Human	Reverse	GTGGATGATTCGGCACTTCAGGAG

表2 各组患者的一般情况比较 (x±s)

Tab 2 Comparison of general conditions of patients in the four groups (x±s)

Group	Subgroup Gestational age/week	Number/n	Age/year	P value^*	P value^#
URPL (n=59)	5?5⁺⁶	20	28.80±4.25		0.987^a
	6?7⁺⁶	22	29.87±4.10	0.708	0.741^b
	8?9⁺⁶	17	29.29±4.31		0.850^c
Induced abortion (n=70)	5?5⁺⁶	22	29.05±3.98
	6?7⁺⁶	27	28.85±3.97	0.913
	8?9⁺⁶	21	29.33±3.62
Normal pregnancy with final live birth (n=157)	5?5⁺⁶	53	28.91±3.81
	6?7⁺⁶	59	29.20±4.23	0.865
	8?9⁺⁶	45	28.78±4.53
Normal pregnancy with subsequent pregnancy loss (n=19)	5?5⁺⁶	8	29.38±4.14
	6?7⁺⁶	11	30.18±4.49	0.695
	8?9⁺⁶	0	?

表3 各组患者的外周血PRL水平（x± s)

Tab 3 PRL levels in peripheral blood of women in four groups （x± s)

Group	Subgroup Gestational age/week	Number/n	PRL/(ng·mL^-1)	P value ^*	P value^#
Group	Subgroup Gestational age/week	Number/n	PRL/(ng·mL^-1)	P value ^*	URPL	Induced abortion	Normal pregnancy with subsequent pregnancy loss
Normal pregnancy with final live birth (n=157)	5?5⁺⁶	53	28.88±11.38	0.002^a	0.876	0.659	0.442
	6?7⁺⁶	59	35.69±11.30	0.012^b	0.018	0.547	0.021
	8?9⁺⁶	45	42.22±14.66	?	0.015	0.113	?
Induced abortion ( n=70)	5?5⁺⁶	22	27.59±11.40	0.015^a	0.622	?	0.660
	6?7⁺⁶	27	37.47±15.11	0.023^b	0.024	?	0.041
	8?9⁺⁶	21	49.06±18.94	?	0.003	?	?
URPL (n=59)	5?5⁺⁶	20	29.34±11.39	0.805^a	?	?	0.412
	6?7⁺⁶	22	28.49±10.79	0.306^b	?	?	0.791
	8?9⁺⁶	17	32.28±11.91	0.450^c	?	?	?
Normal pregnancy with subsequent pregnancy loss (n=19)	5?5⁺⁶	8	25.63±8.33	0.635^a	?	?	?
Normal pregnancy with subsequent pregnancy loss (n=19)	6?7⁺⁶	11	27.52±8.77	?	?	?	?

图2 正常妊娠活产组、人工流产组、URPL组和正常妊娠流产组妇女的外周血PRL水平Note: Normal pregnancy with final live birth group:①P = 0.002, compared with women with the gestation of 6?7+6 week in the same group;②P=0.012, compared with women with the gestation of 8?9+6 week in the same group.Induced abortion group: ③P = 0.015, compared with women with the gestation of 6?7+6 week in the same group; ④P = 0.023,comparedwithwomen with the gestation of 8?9+6 week in the same group. URPL group: ⑤P = 0.018, compared with women innormal pregnancy with final live birth group with the same gestation of 6?7+6 week; ⑥P =0.024, compared with women in the induced abortion group with the same gestation of 6?7+6 week; ⑦P = 0.015, compared with womeninnormal pregnancy with final live birth group with the same gestation of 8?9+6 week; ⑧P =0.003, compared with women ininduced abortion group with the same gestation of 8?9+6 week. Normal pregnancy with subsequent pregnancy loss group: ⑨P = 0.021, compared with women innormal pregnancy with final live birth group with the same gestation of 6?7+6 week; ⑩P = 0.041, compared with women ininduced abortion group with the same gestation of 6?7+6 week.

Fig 2 PRL levels in peripheral blood of women in normal pregnancy with final live birth group, induced abortion group, URPL group and normal pregnancy with subsequent pregnancy loss group

图3 蜕膜基质细胞的鉴定（IHC和IF方法）Note:A / B. IHC of decidua against keratin and vimentin respectively. Black arrow with line indicated glandular epithelium and red arrow indicated DSCs. C. Optical microscopic image of DSCs cultured in vitro. D. IF of DSCs cultured in vitro, keratin (red), vimentin (green), nucleus (blue), DAPI: chromatin dye.

Fig 3 Identification of decidual stromal cells by immunohistochemistry and immunofluorescence

表4 人工流产组和URPL组中PRL在蜕膜基质细胞中的表达

Tab 4 PRL expression of decidual stromal cells in the UPRL and induced abortion groups

Indicator	Group
Indicator	URPL (n=59)			Induced abortion (n=70)
Method of Measurement
Subgroup gestational age/week	5?5⁺⁶	6?7⁺⁶	8?9⁺⁶	5?5⁺⁶	6?7⁺⁶	8?9⁺⁶
Number/n	20	22	17	22	27	21
RT-qPCR (RNA)
PRL (/β-actin/induced abortion)	1.03±0.46	1.13±0.42	1.46±0.39	1.16±0.42	1.49±0.53	1.95±0.65
P value^* (Intra-group comparison)	0.453^a	0.016^b		0.020^a	0.011^b
P value^# (Comparison between groups)	0.336	0.012	0.011
WB (protein)
PRL (/β-actin)	0.23±0.10	0.25±0.11	0.34±0.13	0.24±0.12	0.35±0.17	0.46±0.19
P value* (Intra-group comparison)	0.715^a	0.016 ^b		0.010^a	0.034^b
P value^# (Comparison between groups)	0.865	0.014	0.030
IHC (protein)
PRL (AOD)	0.078±0.039	0.085±0.035	0.113±0.037	0.083±0.043	0.111±0.043	0.145±0.042
P value^*(Intra-group comparison)	0.546^a	0.021^b		0.030^a	0.012^b
P value^# (Comparison between groups)	0.697	0.028	0.026

图4 人工流产组和URPL组中PRL在蜕膜基质细胞中的表达Note: A. Relative mRNA expression of PRL in DSCs detected by RT-qPCR, ①P = 0.020, compared with women of 6?7+6 week with in the same group; ②P = 0.011, compared with women of 8?9+6 week with in the same group; ③P = 0.012, compared with women in the URPL group at the same gestation of 6?7+6 week; ④P= 0.011, compared with women in the URPL group at the same gestation of 8?9+6 week; ⑤P = 0.016, compared with women of 6?7+6 week within the same group. B/C. Expression of PRL protein in the same group at different gestation detected by WB of DSCs. D. Expression of PRL protein between the two groups at the same gestation detected by WB of DSCs. E. Quantitative representation of WB.①P = 0.010, compared with women of 6?7+6 week within the same group; ②P = 0.034, compared with women of 8?9+6 week with in the same group; ③P = 0.014, comparedwith women in URPL group at the same gestation of 6?7+6 week; ④P= 0.030, compared with women in URPL group at the same gestation of 8?9+6 week; ⑤P = 0.016,compared with women of 6?7+6 week with in the same group. F. IHC of the decidua against PRL. G. Quantitative representation of the average optical density (AOD) of PRL in IHC.①P = 0.030, comparedwith women of 6?7+6 week with in the same group; ②P = 0.012, compared with women of 8?9+6 week within the same group; ③P = 0.028, compared with women in URPL group at the same gestation of 6?7+6 week; ④P= 0.026, compared with women in the URPL group at the same gestation of 8?9+6 week; ⑤P= 0.021,compared with women of 6?7+6 week with in the same group.

Fig4 PRL expression of decidual stromal cells in UPRL and induced abortion groups

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