Int J Clin Exp Med 2020;13(5):3355-3362 www.ijcem.com /ISSN:1940-5901/IJCEM0107940 Original Article Placental pathological manifestations and expression levels of apoptosis-related genes and HSP-70 in fetal distress and premature rupture of membranes Yuexia Song1*, Qiaohua Li2*, Zhongting Fan3, Weixia Wang1, Guiping Xu4 1Department of Obstetrics, Laoling People’s Hospital, Laoling, Shandong Province, China; 2Department of Obstet- rics, Linqing Municipal People’s Hospital, Linqing, Shandong Province, China; 3Department of Obstetrics, Weifang Hospital of Maternal and Child Health, Weifang, Shandong Province, China; 4Department of Obstetrics, Zibo City Linzi District People’s Hospital, Zibo, Shandong Province, China. *Equal contributors and co-first authors. Received January 15, 2020; Accepted March 3, 2020; Epub May 15, 2020; Published May 30, 2020 Abstract: Objective: The present study was designed to investigate the pathological manifestations, cell apoptosis and the expression of heat shock protein (HSP)-70 in postpartum placenta of patients with fetal distress or pre- mature rupture of membranes (PROM). Methods: The pathological changes, apoptosis and HSP-70 expression of placenta in 35 cases of fetal distress, 40 cases of premature rupture of membranes and 30 cases of normal de- livery were retrospectively analyzed. Results: Compared with the normal placenta group, the placental pathological manifestations of the fetal distress group and the PROM group showed increased apoptosis of villous trophoblasts and decidual cells (P<0.001). Reverse transcription PCR (RT-PCR) revealed that the mRNA levels of apoptotic genes CDKN1A and BAX were significantly elevated (P<0.001). The expression level of HSP-70 in cells was also found to be markedly increased as measured by Western blot (P<0.01). Conclusion: Increased placental cell apoptosis may be involved in the pathological changes of fetal distress and PROM, in which the expression of apoptosis-related genes CDKN1A, BAX, and HSP-70 is also increased significantly. Keywords: Fetal distress, premature rupture of membranes, pathological manifestations, apoptosis, heat shock protein-70 Introduction vitro environment are also implicated in the occurrence of the diseases [2]. Placenta is an Fetal distress and premature rupture of mem- important bridge between the infant and the branes (PROM) are common complications in mother. Pathological observation of placental the second and third trimester of pregnancy. tissues after delivery has revealed that the According to the statistics, the two account for occurrence of a variety of gestational disea- 1.5-5.6% among all the complications in the ses is related to the abnormal number and middle and terminal pregnancy, resulting in a function of villous trophoblast cells and deci- neonatal mortality of 0.5-0.9%. Improper treat- dual cells [3], as well as the increase of cell ment will seriously affect the outcome of preg- apoptosis. Apoptosis is one of the ways of pro- nancy and the safety of mother and baby. Al- grammed cell death. Changes in the external though fetal distress and PROM are two inde- environment activate apoptosis-related coordi- pendent diseases, recent studies have found nation and receptor binding, which in turn initi- that increased apoptosis of placental tissues ates the transcription and translation of apop- caused by different degrees of immune rejec- tosis-related genes, and participates in the ex- tion between the placenta and the mother may pression of various apoptotic proteins and ac- be an important mechanism for the occurren- tive factors, such as CDKN1A, BAX, and heat ce of these diseases [1]. In addition, genetic shock protein-70 (HSP)-70 [4-6]. defects, anatomical abnormalities of the re- productive system, endocrine disorders, infec- Increased placental cell apoptosis not only ex- tions, thrombus and abnormal in vivo and in ists in the placental tissues of pregnant wo- Postpartum placenta of pregnant women with fetal distress or PROM men with abnormal pregnancy, but also can nital abnormalities in fetus; 3. Patients with be observed in the physiological processes of severe liver and kidney dysfunction. trophoblast cell apoptosis, blastocyst implan- tation and growth, degeneration and recon- Research methods struction of decidual tissues, as well as struc- tural remodeling of placenta. Furthermore, After admission, patients with fetal distress with the continuous increase of gestational or PROM actively cooperated to complete re- weeks, there are different pathological signs, levant examinations, closely monitored fetal and placental cell proliferation and apoptosis intrauterine growth and labor progress, and are often in a dynamic equilibrium. Once the tried to extend the gestational week as far as apoptotic activity is higher than the prolifera- possible. Whereas, the pregnancy was termi- tion, it may trigger a variety of pregnancy-relat- nated immediately in case the delivery condi- ed diseases [7]. What’s more, the expression tions were appropriate or the risk of maternal activity of HSP-70 is enhanced in the hypoxic and infant complications was high. According environment of cells, thus inducing cell dama- to the delivery conditions, natural delivery or ge repair, apoptosis and other adaptation to cesarean section was properly selected to environmental changes. HSP-70 is highly con- ensure the complete delivery of the placenta. served and mainly plays the role of “molecular The pathological manifestations of the placen- chaperone”, which is bound up with cell prolif- ta were observed under a light microscope, eration and apoptosis. It has been found in in the apoptosis rate of placental cells was de- vitro culture of various tumor cells that HSP-70 tected by TUNEL method, the mRNA express- can significantly facilitate cell apoptosis and ion levels of apoptosis gene CDKN1A and BAX inhibit cell proliferation under hypoxia environ- were measured by reverse transcription PCR ment. Based on this, the main purpose of this (RT-PCR), and the expression of HSP-70 was study was to explore the pathological manifes- determined by Western blot. As soon as the tations, apoptosis and HSP-70 expression of placenta was successfully delivered, it was placenta after delivery in patients with fetal dis- fixed in 10% neutral formalin solution to pre- tress or PROM. pare paraffin-embedded serial sections with a thickness of 3 μm and then placed in an oven Research paricipants and methods at 70°C for 3 hours. Data of research participants Detection of apoptosis rate by TUNEL method In this study, 35 cases of fetal distress, 40 cases of PROM, and 30 cases of normal preg- Main steps: The placenta was pretreated with nancy admitted to our hospital from August paraffin sections, followed by dewaxing, hy- 2018 to August 2019 were enrolled for a re- dration and PBS washing. Then, the protease trospective analysis. Inclusion criteria: 1. Pati- K solution (20 μg/mL, Sangon Biotechnology ents who met the diagnostic criteria for fetal Co., Ltd., Shanghai, China) was added drop- distress and PROM based on the 9th edition wise, and then 100 μL of it was incubated for of Obstetrics and Gynecology [8], those with 8 minutes at room temperature. On this basis, normal pregnancy and without assisted repro- anti-fluorescein and TUNEL marker solution duction; 2. All were pregnant with single live were added dropwise, and then washed 3 births, no twins or multiple pregnancies; 3. All times with PBS, 5 minutes each. After equili- were delivered successfully and the mother brating with buffer solution III, the color was and baby were discharged safely; 4. The pla- developed at room temperature, and the nitro- centa was delivered intact after the fetus was blue tetrazolium/bromochloroindolyl phospha- delivered, and placental tissue sections could te was added dropwise to observe the color. be prepared for subsequent research; 5. Infor- med consent was obtained and clinical data RT-PCR detection of CDKN1A and BAX mRNA were complete. Exclusion criteria: 1. Patients levels with pregnancy complications, such as pre- eclampsia, gestational diabetes, placenta pre- The main steps were as follows: Firstly, the via, or amniotic fluid abnormalities; 2. Conge- placental tissue cells were smashed by ultra- 3356 Int J Clin Exp Med 2020;13(5):3355-3362 Postpartum placenta of pregnant women with fetal distress or PROM Table 1. Target primer sequence and size Primer name Forward sequence Reverse sequence Size (bp) CDKN1A 5’-GCAGCGGAACAAGGAGT-3’ 5’-GGAGAAACGGGAACCAG-3’ 256 BAX 5’-CCCCCGAGAGGTCTTTTTCC-3’ 5’-TGTCCAGCCCATGATGGTTC-3’ 165 GAPDH 5’-GGCTACAGCAACAGGGTG-3’ 5’-TTTGGTTGAGCACAGGGT-3’ 148 Table 2. Comparison of baseline data of three groups of pregnant women and fetuses Group Fetal distress Premature rupture of membranes Normal pregnancy F/χ2 P Number of cases 35 40 30 Age (age) 28.6±5.5 29.2±6.3 27.7±5.3 0.639 0.258 Body mass index (kg/m2) 26.5±3.2 26.8±3.4 26.7±3.5 0.076 0.927 Primiparous/menopausal 20/15 25/15 17/13 0.320 0.852 Gestational week (week) 37.8±1.3 37.9±1.5 37.5±1.2 0.432 0.502 Abortion/cesarean section 18/17 23/17 16/14 0.293 0.864 Baby/baby girl 17/18 21/19 14/16 0.252 0.881 Birth weight (kg) 3.5±0.6 3.4±0.5 3.6±0.7 0.735 0.169 sound and washed and dissolved by PBS solu- electrophoresis before the charged separation tion. Then, RNA molecules were extracted from area was transferred to PVDF membrane, fol- placental cells by one-step method according lowed by the dropwise addition of rat anti- to TRIzol kit (Invitrogen Company, USA). After human HSP-70 and β-actin primary antibody determining the concentration and purity of (1:2000, sigma company of America) in sequ- RNA, cDNA was synthesized by reverse tran- ence and left to stand overnight. After wash- scription kit (Dalian TaKaRa, China). The target ing with PBS, rabbit anti-rat secondary anti- primer sequence was synthesized by Takara body (1:500, sigma, USA) was added and in- Company, Japan, with reference to Gene Bank cubated at room temperature for 4 h, washed (Table 1).