4344 DOI 10.1002/pmic.200800294 Proteomics 2008, 8, 4344–4356 RESEARCH ARTICLE Comparative proteomic analysis of human placenta derived from assisted reproductive technology Yu Zhang1*, Yan-Ling Zhang1, 2*, Chun Feng1, Yan-Ting Wu1, Ai-Xia Liu1, Jian-Zhong Sheng3, Jie Cai1 and He-Feng Huang1, 2 1 Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 2 Key Laboratory of Women’s Reproductive Health, Hangzhou, China 3 Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Canada The aim of this study was to use proteomics-based approach to examine differences in protein Received: April 4, 2008 expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Revised: May 19, 2008 Using 2-DE we found that, compared with the control group, 12 spots in standard in vitro fertilization Accepted: June 10, 2008 group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differ- entially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially ex- pressed in both ARTgroups were identified as annexin A3, hnRNP C1/C2, a-SNAP, FTL and ATP5A. Some ofthe proteinswere confirmed by Western blot and immunohistochemistry analysis.Ourstudy allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ARTplacenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome. Keywords: 2-DE / Annexin A3 / Assisted reproductive technology / MALDI-TOF/TOF / Placenta 1 Introduction However, in recent years, more and more well-designed studies have consistently documented associations between ART and Assisted reproductive technology (ART), containing standard in an increased risk of birth defects [1, 2], low birth weight [3], vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) chromosome abnormalities [4, 5] and possibly, childhood can- and their related procedures, have helped thousands of couples cer. These results were also confirmed in a number of system- conceive children in the past 30 years. Early studies suggested atic reviews [6, 7]. In addition, a possible link has now been ARTwas safe and children born from ARTdeveloped normally. suggested between ARTand epigenetic defects. Several clinical studies reported an increased frequency of imprinting defects Correspondence: Professor He-Feng Huang, Department of Repro- among ART children [8, 9]. Although the precise significance ductive Endocrinology, Women’s Hospital, School of Medicine, and origin of these associations require confirmation and clar- Zhejiang University, 2 Xue Shi Road, Hangzhou, Zhejiang, 310006, ification, available evidence has suggested the concerns for the China long-term safety of ARTprocedures. E-mail: [email protected] As IVF was introduced into practice without formal evalua- Fax: 186-571-8706-1878 tion of its effects on the health of the children conceived with this procedure [1], there is little information about the mechan- Abbreviations: ART, assisted reproductive technology; ICSI, intracy- toplasmic sperm injection; IHC, immunohistochemistry; IVF, in vitro fertilization; IVP, in vitro embryo production; RT,roomtemperature * These authors contributed equally to this work. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2008, 8, 4344–4356 Biomedicine 4345 isms underlying the increased health defects. It is also unclear the period of April 2006 to May 2007 in Women’s Hospital, at which stage (s) from the controlled ovarian hyperstimulation, School of Medicine, Zhejiang University, China. We had gametes manipulation, in vitro culture, embryo transfer to the access to complete clinical data for each individual. The baby’s birth, the risk may be raised. Since ICSI was introduced inclusion criteria used for ART patients were as follows: for male infertility, more concerns were also raised that ICSI maternal age between 25 to 35 years; full-term delivery, sin- would increase the risk of birth defects and genetic disorders as gleton pregnancy, child birth weight between 2500 to 4000 g, it bypasses almost all the selection mechanisms that operate in no indication of pregnancy complications and no birth natural conception and the invasive procedure of ICSI may have defect. Couples referred for ART because of maternal oviduct a deleterious effect [10]. Moreover, possible causes leading to obstruction for standard IVF group and male infertility (oli- abnormalities of offspring derived from ART are much more gospermia and/or asthenozoospermia) with or without complicated. Generally, it is difficult to determine whether birth maternal oviduct problem for ICSI group. The control group defects after assisted reproduction result directly from the pro- was formed by age-matched women with natural, singleton cedures used, or whether they reflect the suboptimal fertility of pregnancy and underwent caesarean surgery without suffer- the parents. To determine whether ART is associated with ing any disease during pregnancy. The clinical application of health defects and what is the mechanism for this association, ART was licensed by the Health Ministry of People’s Repub- additional in-depth investigations are required. lic of China. The study was approved by the Ethical Com- The placenta is a temporary organ that consists of a fetal mittee of Women’s Hospital, School of Medicine, Zhejiang component and a maternal component and it is a structure University and informed written consent was obtained from unique to pregnancy functioning to sustain and protect the each woman for the use of placenta. fetus until birth. The placenta also serves as an endocrine organ producing a wide range of important signals that ensure the 2.2 Placenta sample collection maintenance of gestation and fetal well-being. Abnormal pla- centation has been observed following in vitro embryo produc- The placenta was collected and dissected immediately fol- tion (IVP) in animals [11, 12]. It is also suggested that heavy fetal lowing delivery. Fragments from the placental subchorial losses and abnormalities in animal somatic cell nuclear transfer zone corresponding to umbilical cord insertion were dis- might be associated with abnormal placenta development par- sected. Maternal membranes were eliminated, and floating tially because of embryo manipulation and in vitro culture [13, villi were washed in ice-cold PBS. Placental samples were 14]. Meanwhile, it has been reported that human placenta stored in liquid nitrogen just after collection for half an hour derived from ARTare associated with more frequent pathologi- and finally stored at 2807C until further protein extraction. cal findings [15]. These results of abnormal placentation in assisted reproduction related procedures in animals and hu- 2.3 Protein preparation man being reveal partial evidence for the possibility of worse outcome of ARToffspring. To overall evaluate the specific gene For experiments, a total of 300 mg of each placental villous products in placenta following ART, global screening of protein sample was homogenized and solubilized in 1000 mL of lysis expression in placenta is needed. buffer containing 7 M urea, 2 M thiourea, 4% w/v CHAPS, Proteomics, the large-scale study of proteins, is the cur- 1% w/v DTT, 2% v/v IPG buffer (pH 4–-7), 1% v/v PMSF and rently most powerful technique for protein identification in 1% v/v protease inhibitor cocktail and kept on ice for 1 h. complex cellular systems. High-resolution 2-DE in combi- Sonication was carried out on ice at a power of 90 W for five nation with MS is an excellent tool for proteomic analysis. It bursts of 8 s, each interspersed with 16 s. Then, the lysed cells gives information in a high-throughput mode about protein were centrifuged at 15 000 6 g at 47C for 60 min to remove expression profiles. Thus, proteomics may enable us to debris. Protein concentrations were determined using the unravel particular molecular complexes or pathways in the Bradford protein assay (Bio-Rad) [16]. The final protein sam- pathogenesis of the human placentation following ART. In ples were stored at 2807C prior to electrophoresis. the present study, we analyzed differential protein patterns between placentas obtained from ART (standard IVF or 2.4 2-DE ICSI) and normal placenta obtained from natural conception using 2-DE and MS to find potential effects of ART on the The 2-DE was performed using the Protean IEF Cell and expression of proteins in placenta. Protean Plus Dodeca Cell (Bio-Rad, USA) according to the manufacturer’s instructions. For the first dimension, IEF was performed on Protean IEF Cell with ReadyStrip IPG 2 Materials and methods strips (Linear, pH 4–7, 24 cm, Bio-Rad). 400 mg of total pro- tein was mixed with a rehydration solution containing 7 M 2.1 Subjects and ethics urea, 2 M thiourea, 2% w/v CHAPS, 0.4% w/v DTT, 0.5% IPG buffer (pH 4–7) and 0.001% bromophenol blue to a total Placentas from ART (standard IVF and ICSI) and normal volume of 450 mL. Following IEF separation, the strips were pregnancies were collected after caesarean delivery during incubated for 15 min in an equilibration buffer (6 M urea, © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 4346 Y. Zhang et al. Proteomics 2008, 8, 4344–4356 50 mM Tris-HCl pH 8.8, 30% v/v glycerol, 2% SDS, and rate with wavelength of 355 nm. The accelerating voltage was 0.001% bromophenol blue) containing 1% DTT and subse- operated at 20 kV. Myoglobin digested by trypsin was used to quently in the same equilibration buffer containing 2.5% calibrate the mass instrument with internal calibration mode.