Association between polymorphisms of prokineticin receptor (PKR1 rs4627609 and PKR2 rs6053283) and recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a condition with complex etiologies, to which both genetic and envi- ronmental factors may contribute. During the last decade, studies indicated that the expression patterns of the pro- kineticin receptor (PKR1 and PKR2) are closely related to early pregnancy. However, there are few studies on the role of PKR1 and PKR2 in RPL. In this study, we purpose to investigate the association between polymorphisms of the prokineticin receptor (PKR1 rs4627609 and PKR2 rs6053283) and RPL on a group of 93 RPL cases and 169 healthy controls. Genotyping of the single nucleotide polymorphisms (SNPs) was performed using a Sequenom MassARRAY iPLEX system. The results revealed a significant asso ciation between PKR2 rs6053283 polymorphism and RPL (P=0.003), whereas no association was observed between PKR1 rs4627609 polymorphism and RPL (P=0.929) in the Chinese Han population. Key words: Prokineticin receptor 1 (PKR1), PKR2, Polymorphism, Recurrent pregnancy loss http://dx.doi.org/10.1631/jzus.B1500180 CLC number: R714.21 1 Introduction Recurrent pregnancy loss (RPL) is defined as resulting in at least two consecutive idiopathic mis- carriages before 20 weeks of gestation by the Practice Committee of the American Society for Reproductive Medicine (2013). Approximately 2%–4% of the women of childbearing age are suffering from this problem (Li et al., 2001). The causes of RPL are complicated: chromosome abnormalities, anatomical abnormalities, endocrinology disorders, immune disorders, thrombophilia, maternal infections, and nutritional deficiencies whicn are the most common etiologies; however, 50% of RPLs with no obvious causes are known as unexplained RPLs (Li et al ., 2002). Prokineticins (PKs) are recently discovered to be tissue-specific angiogenesis factors, comprising of PK1 and PK2, which are also known as endocrine gland-derived vascular endothelial growth factors (EG-VEGFs) and Bv8. In humans, EG-VEGF and Bv8 with 83% amino-acid identity, play the same role and possess the same G protein-coupled receptors, and are defined as prokineticin receptor 1 (PKR1) and PKR2 (Lin et al., 2002; Le Couter et al., 2003). PK1 expression is primarily localized in the steroidogenic glands including the ovary, testis, adrenal gland, and Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) ISSN 1673-1581 (Print); ISSN 1862-1783 (Online) www.zju.edu.cn/jzus; www.springerlink.com E-mail: [email protected] ‡ Corresponding authors * Project supported by the National Natural Science Foundation of China (No. 81571503) ORCID: Jing DU, http://orcid.org/0000-0002-6767-2763 © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 Cao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(3):218-224 219 placenta (Le Couter et al., 2001). The distribution of PK2 expression is similar to PK1, but is found to be at the highest level in testis and peripheral blood leu- kocytes (Li et al., 2001). Le Couter et al. (2001) have briefly reported PK1 expression in the placenta. Hoffmann et al . (2006) have recently characterized PK1 expression in human placenta during the early period of pregnancy and suggested that PK1, but not PK2, expresses in human placenta and is primarily located in the syncytiotrophoblast layer, during the 8 to 10 weeks of gestation, and the expression of PK1 emerges as the highest level and can be up-regulated by hypoxia. PK1 can promote angiogenesis via in- ducing tissue-specific proliferation, migration and fenestration in capillary endothelial cells, and the function of angiogenesis seems to be limited to en- dothelial cells derived from endocrine tissues (Cheng et al ., 2002). Furthermore, microarray analysis re- vealed that PK1 may participate in regulating the expression of multiple cytokines and implantation of endometrium (Evans et al., 2008; Haouzi et al., 2009). PKs play a role through their receptors (PKRs). As a

of binding with PKs, PKRs couple to Gq pro- teins, and then promote intracellular Ca 2+ mobiliza- tion (Lin et al., 2002; Soga et al., 2002). In addition, PKRs can also couple to Gi and Gs proteins, indi- cating that PKRs are found to be involved in activat- ing multiple intracellular signal-transduction path- ways (Chen et al ., 2005). Activation of PKRs influ- ences several physiological events, such as intestinal contraction, spermatogenesis, circadian rhythm, hematopoiesis, and angiogenesis (Schweitz et al., 1999; Wechselberger et al ., 1999; Le Couter et al ., 2001; 2002; 2003; 2004; Morton et al., 2005). Given the potential regulation of the PKs/PKRs pathways in human early pregnancies, we hypothe- sized that polymorphisms of PKRs are involved in abnormal angiogenesis and implantation failure. The present study aims to inve stigate the association be- tween the polymorphisms of PKRs (PKR1 rs4627609 and PKR2 rs6053283) and RPL. 2 Materials and methods 2.1 Subjects The study was approved by the Ethics Commit- tee of Soochow University (Suzhou, China). A total of 93 RPL cases (mean±standard deviation (SD) age, (28.30±3.77) years; body mass index (BMI), (20.93± 2.23) kg/m 2) and 169 ethnic-matched healthy controls (mean±SD age, (28.08±3.62) years; BMI, (20.10± 2.29) kg/m 2) were included in this study. Patients aged 20–40 years with two or more consecutive un- explained terminations of pregnancy before 12 gesta- tional weeks or expulsions of a fetus weighing <500 g were identified and recruited when they visited the Maternal and Child Health Center in Kunshan City, the First People’s Hospital and the Second People’s Hospital which are affiliated with Soochow University for repeated treatment (two or more times). All pa- tients with RPL had a naturally conceived pregnancy. Subjects with anatomical abnormality, chromosome abnormality, endocrine disorder, autoimmune disease, or coagulopathy were excluded from the study. One hundred and sixty-nine age-matched pregnant women with at least one naturally conceived pregnancy and no history of miscarriage were recruited as control subjects (2 for each case patient). None of the controls had a history of pregnancy complications, still birth, pre-eclampsia, or giving birth to a child who was small for its gestational age. The matching criteria included age (±2 years), gestational age (±1 week), and residence (the same district). Information on demographic characteristics, lifestyle, obstetric history, and other RPL risk factors was collected using a structured questionnaire. An- ticoagulant peripheral blood samples were obtained from all the participants during the following two weeks after their day of recruitment, and stored at −80 °C until the genomic DNA was isolated. 2.2 Genotyping Genomic DNA was extracted from the lym- phocyte of the anticoagulant peripheral blood using the QIA-amp DNA blood kit (QIAGEN, Hilden, Germany) according to the manufactures’ instructions. The quality evaluation of the extracted DNA was performed using a NanoDrop spectrophotometer (ThermoScientific) by measuring the absorbance at 260 nm. The DNA sample used for genotyping is 30 ng/ μl with an OD 260/OD280 (OD: optical density) range from 1.8 to 2.0 and an OD260/OD230 range of no less than 1.5. The single nucleotide polymorphisms (SNPs) of rs4627609 and rs6053283 were detected by using a Sequenom MassARRAY iPLEX platform (Sequenom, San Diego, CA, USA), in which a 90% sample quality Cao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(3):218-224 220 control (QC) rate and a 90% SNP genotyping success rate were imposed. Briefly, a single base extension, following an initial locus-specific PCR reaction, was conducted by using mass-modified dideoxynucleotide terminators of an oligonucleotide primer which im- mediately anneals upstream to the polymorphic site of interest, and then, via using a matrix-assisted laser desorption and ionization time-of-flight (MALDI- TOF) mass spectrometry, the SNP allele was identi- fied by the distinct mass of the extended primer (Ga- briel et al., 2009). 2.3 Statistical analysis The Hardy-Weinberg equilibrium and the com- parisons of genotype and allele frequencies between RPL cases and the controls were performed using the online software SHEsis to perform a Chi-square test (http://analysis.bio-x.cn/myAnalysis.php) (Shi and He, 2005). Using the SPSS Version 13.0, a univariate logistic regression was conducted, and then adjusted by the BMI of the participants. The independent- samples t-test between the RPL cases and controls on the women’s age and BMI showed that there was a significant difference between the case and control groups on BMI ( P=0.005; Table 1). The adjusted odds ratios (AORs) and their 95% confidence inter- vals (CIs) were used to measure the strength of the association between the RPL and polymorphisms of rs4627609 and rs6053283. In order to account for multiple testing, significance levels were adjusted by the Bonferroni correction, which was made for 2 SNPs, and the significance level was reduced to 0.025 (0.05/2), where appropriate (O'Callaghan et al., 2012). 2.4 Prediction of SNP effects The prediction of the SNP effects on mRNA splicing was performed using the web-based predic- tion software ESEfinder (http://rulai.cshl.edu/cgi-bin/ tools/ESE3/esefinder.cgi?process=home) (Smith et al., 2006). 3 Results The genotype distributions of the two poly- morphisms both in the case and control groups were all within the Hardy-Weinberg equilibrium (P>0.05). The data of genotype and allele frequencies are shown in Tables 2 and 3. The results indicated that the dis- tribution of the genotype and allele frequencies of PKR2 rs6053283 showed significant differences be- tween the RPL cases and the healthy controls (P=0.009 and P=0.003, respectively). The univariate logistic re- gression analyses showed that women with rs6053283 AA genotype had a higher risk of RPL than GG gen- otype carriers (AOR: 2.65, 95% CI: 1.14–6.17, P=0.024) and A allele was positively associated with the in- creased risk of RPL (AOR: 2.14, 95% CI: 1.28–3.58, Table 1 Independent-samples t-test between case and control subjects Group Age (year) BMI (kg/m 2) Case (n=93) 28.301±3.767 20.926±2.226 Control (n=169) 28.083±3.618 20.103±2.289 P-value 0.650 0.005 Data are expressed as mean±SD. Age: age of the participants; BMI: body mass index of the participants Table 2 Genotype and allele frequencies of the PKR1 rs4627609 SNP in RPL patients and controls SNP Group Genotype frequency Allele frequency TT TC CC P-value T C P-value OR (95% CI) PKR1 (rs4627609) Case 38 (0.409) 37 (0.398) 18 (0.194) 0.714 113 (0.608) 73 (0.392) 0.929 0.98 (0.68–1.42) Control 64 (0.379) 76 (0.450) 29 (0.172) 204 (0.604) 134 (0.396) Data are expressed as number (frequency) Table 3 Genotype and allele frequencies of the PKR2 rs6053283 SNP in RPL patients and controls SNP Group Genotype frequency Allele frequency GG GA AA P-value G A P-value OR (95% CI) PKR2 (rs6053283) Case 63 (0.677) 25 (0.269) 5 (0.054) 0.009 151 (0.812) 35 (0.188) 0.003 2.14 (1.28–3.58) Control 137 (0.811) 31 (0.183) 1 (0.006) 305 (0.902) 33 (0.098) Data are expressed as number (frequency) Cao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(3):218-224 221 P=0.003) (Table 4); however the polymorphism of rs4627609 was not associated with RPL. The wild-type (G) allele and mutant-type (A) allele of PKR2 rs6053283 were analyzed using ESEfinder, and the data indicated that the mutant-type “A” allele can affect the splicing regulation by alter- ing the exonic splicing enhancer (ESE) motifs. The

showed that the mutant-type “A” results in a gain within the SC35 motif (1.12) (Fig. 1). Serine/ arginine-rich (SR) proteins, which have distinct func- tions in alternative pre-mRNA splicing, are bound to ESEs and function as general activators of the exon definition. The SR protein family members include SF2/ASF, SC35, SRp20, SRp40, SRp55, SRp75, SRp30c, 9G8, and SRp54 (Long and Caceres, 2009). 4 Discussion The results of the independent-samples t-test between the RPL case and the control groups on women’s ages and BMI revealed that there were sig- nificant differences between the case and control groups in BMI ( P=0.005). In order to reduce the in- fluence of the confounding factors, the OR was ad- justed by the BMI for all the subjects. Although, the AOR was estimated as a measure of the strength of the association between the polymorphisms and RPL, it was still difficult to rule out other confounding factors, such as the living environments and dietary habits. Even so, the results demonstrated a strong association between the polymorphism of PKR2 rs6053283 and RPL (P=0.003). The beginning and maintenance of a successful pregnancy depends on many factors, such as angio- genesis and endometrial receptivity. Angiogenesis is very important in the process of placental vessel formation and maternal vascular adaptation. PK1 and its receptors (PKR1, PKR2), are shown to be expressing in the human placenta of the first and third Table 4 Univariate logistic regression for dependent variables: having RPL according to genotype distributions of investigated SNPs as independent variables Genotype and allele Number (frequency) AOR (95% CI) P-value Case (n=93) Control ( n=169) PKR1 rs4627609 TT 38 (0.409) 64 (0.379) 1.00 (ref) TC 37 (0.398) 76 (0.450) 1.20 (0.68–2.14) 0.527 CC 18 (0.194) 29 (0.172) 0.94 (0.66–1.36) 0.746 TC+CC 1.10 (0.65–1.87) 0.718 T 113 (0.608) 204 (0.604) 1.00 (ref) C 73 (0.392) 134 (0.396) 0.98 (0.68–1.42) 0.929 PKR2 rs6053283 GG 63 (0.677) 137 (0.811) 1.00 (ref) GA 25 (0.269) 31 (0.183) 1.64 (0.88–3.06) # 0.122 AA 5 (0.054) 1 (0.006) 2.65 (1.14–6.17) 0.024 GA+AA 1.94 (1.07–3.50) 0.028 G 151 (0.812) 305 (0.902) 1.00 (ref) A 35 (0.188) 33 (0.098) 2.14 (1.28–3.58) 0.003 AOR: adjusted odd ratio by body mass index of female participants; ref: reference group. # Significance level α=0.025 by Bonferroni correction Fig. 1 Prediction for binding of splicing enhancer: an outcome of rs6053283 mutation The wild-type “G” and mutant-type “A” alleles of rs6053283 were predicted by ESEfinder to disrupt ESE sites of one or more of four different SR proteins (SF2/ASF (red), SC35 (blue), SRp55 (yellow)), and the results are shown in the top panels. Colored bars represent individual ESE sites with the matrix score (higher score represents a stronger prediction) plotted on the vertical axis and the position of the ESE within each rs6053283 represented on the horizontal axis (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article) Cao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(3):218-224 222 trimester periods and locating to the syncytiotropho- blast layer, cytotrophoblast layer, fetal endothelium, and macrophages, which mediates tissue-specific angiogenesis, immune regulation, and modulation of the inflammatory responses (Le Couter et al., 2001; Dorsch et al., 2005; Hoffmann et al., 2006; Denison et al., 2008). All of the above functions play critical roles in endometrial receptivity, embryo implantation, and placentation. Meanwhile, the expression pattern of PK1 and its receptor indicates their essential roles in early human pregnancy. The expression of PK1 and PKR1 reaches its peak during the hypoxic period of the placental development. Reports suggested that the dynamic expression of PK1 during the pregnancy period may possibly be regulated by up-regulated hormones during early pregnancy (estrogen, pro- gesterone, and human chorionic gonadotrophin) (Battersby et al., 2004; Evans et al., 2009; Shaw et al., 2010). PK1 and VEGF have similar biological func- tions during human pregnancy (Le Couter et al ., 2003). Both of them are expressed in human placenta; the difference lies in the fact that the VEGF expres- sion is restricted to the cytotrophoblasts and extravil- lous trophoblasts (Hoffmann et al ., 2006), and pol- ymorphisms of the VEGF have been associated with the risk of RPL (Lee et al., 2010). Given these facts, we speculated that PK1 and VEGF perform com- plementary roles during pregnancy. Evidence shows that PK1 expression is elevated during the implantation period (Evans et al., 2008). PK1 was shown to stimulate a group of genes, in- cluding interleukin 11 ( IL-11), IL-6, leukemia inhib- itory factor (LIF), and cyclooxygenase 2 ( COX-2), which play important roles in embryo implantation and decidualization (Stewart et al., 1992; Tabibzadeh et al ., 1995; Lim et al ., 1997; Robb et al ., 1998; Denison et al., 2008; Haouzi et al., 2009). PK1 can induce its target genes by phosphorylation of their downstream target extracellular signal-regulated kinase1/2 through cross-talk with the epidermal growth factor receptor system, or by activation of mitogen- activated protein kinase and the PI3 kinase/Akt pathway (Lin et al., 2002; Haouzi et al., 2009). Den- ison et al. (2008) suggested that PK1 facilitated the embryo implantation via inducing the LIF expression. Recently, PK1 was reported as a uterine receptivity marker (Evans et al ., 2008). All of the evidence unanimously revealed the important role of PK1 in embryo implantation. Therefore, we surmised that the polymorphisms of PK1 and its receptors may be as- sociated with RPL. The mutation of PKR2 has been reported to be associ ated with hypogonadotropic hypogonadism. Kallmann syndrome patients with biallelic PKR2 mutations had a severe reproductive phenotype. Bi- allelic PKR2 mutations have also been reported in women suffering from primary amenorrhea and breast development dysfunction, especially in mono- allelic mutation of PKR2 carriers (Monnier et al ., 2009; Sarfati et al., 2010). Other disorders associated with the PK1 signaling pathway include polycystic ovarian syndrome (PCOS), ectopic pregnancy, en- dometriosis, and pre-eclampsia (Hoffmann et al ., 2006; Shaw et al., 2010; Tiberi et al., 2010). Studies have demonstrated that the PK1 expression is higher in ectopic endometrial and polycystic ovarian tissue in patients with endometriosis or PCOS, and this suggests the role of angiogenesis of PK1 (Ferrara et al., 2003; Shaw et al., 2010). The association be- tween polymorphisms of PK1 and its receptors with RPL was first studied in the Taiwanese Han popula- tion. Su et al. (2010) found that PKR1 rs4627609 and PKR2 rs6053283 polymorphisms were associated with idiopathic RPL. This study was conducted in the East China Han population, and further confirmed the association between PKR2 rs6053283 and RPL. However, we failed to discover the association be- tween PKR1 rs4627609 and RPL. This inconsistent finding may primarily be attributed to the small samples both in our study and previous reports. Moreover, the differences of geographic environ- ments between Taiwanese and East China may also have an influence on the results. To a certain extent, environmental factors play a key role in the occur- rence of RPL as well. In conclusion, our findings indicated that poly- morphism in PKR2 rs6053283 was obviously asso- ciated with idiopathic RPL and could be developed as a new biomarker to evaluate the risk of RPL. How- ever, there is no study performed in other populations and the underlying mechanism of PKR2 rs6053283 polymorphism in the development of RPL is largely unknown, so further studies are warranted to replicate our findings in different ethnic groups with larger sample sizes. It is also necessary to validate the func- tion of the PKR2 rs6053283 polymorphism on RPL. Cao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(3):218-224 223

We would like to thank all the participants for providing their blood samples. This stud y was conducted at the NPFPC Laboratory of Contraception and Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China. Compliance with ethics guidelines Yun-lei CAO, Zhao-feng ZHANG, Jian WANG, Mao-hua MIAO, Jian-hua XU, Yue-ping SHEN, Ai-min CHEN, Jing DU, and Wei YUAN declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Hel- sinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study.

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Steril., 93(7):2145-2149. http://dx.doi.org/10.1016/j.fertnstert.2009.01.105 Wechselberger, C., Puglisi, R., Engel, E., et al ., 1999. The mammalian homologues of frog Bv8 are mainly ex- pressed in spermatocytes. FEBS Lett., 462(1-2) :177-181. http://dx.doi.org/10.1016/S0014-5793(99)01473-8 中文概要 题 目：前动力蛋白受体（PKR1 rs4627609 和 PKR2 rs6053283）的多态性与复发性流产的关系 目的 ： 研究前动力蛋白受体 PKR1 rs4627609 和 PKR2 rs6053283 的多态性与复发性流产的关系，并对 其可能的作用机制进行预测。 创新点：首次在中国汉族人群中进行前动力蛋白受体 PKR1 rs4627609 和 PKR2 rs6053283 的多态性与 复发性流产关系的研究，并对其功能进行预测。 方法 ： 共收集了 93 例复发性流产和 169 例健康对照者 血液样本， 提取基因组DNA， 对PKR1 rs4627609 和 PKR2 rs6053283 两个位点进行基因多态性分 析， 在两组样本中分析不同基因型与复发性流产 的关系，并对 PKR2 rs6053283 位点的不同等位 基因进行生物功能预测。 结论 ： PKR2 rs6053283的多态性与复发性流产相关；而 PKR1 rs4627609的多态性与复发性流产之间不存 在相关性。 关键词：前动力蛋白受体 1；前动力蛋白受体 2；多态性； 复发性流产