{"paper_id":"9668490c-2338-45e5-8ee0-eeea7be465b8","body_text":"TP53 PIN3 and PEX4 polymorphisms and infertility\nassociated with endometriosis or with post- in vitro\nfertilization implantation failure\nDD Paskulin1,3,6, JSL Cunha-Filho 2, CAB Souza 2, MC Bortolini 1,3, P Hainaut 4 and P Ashton-Prolla* ,1,3,5,6\np53 has a crucial role in human fertility by regulating the expression of leukemia inhibitory factor (LIF), a secreted cytokine\ncritical for blastocyst implantation. To examine whether TP53 polymorphisms may be involved with in vitro fertilization (IVF)\nfailure and endometriosis (END), we have assessed the associations betweenTP53 polymorphism in intron 2 (PIN2; G/C, intron 2),\nPIN3 (one (N, non-duplicated) or two (D, duplicated) repeats of a 16-bp motif, intron 3) and polymorphism in exon 4 (PEX4; C/G,\np.P72R, exon 4) in 98 women with END and 115 women with post-IVF failure. In addition, 134 fertile women and 300 women\nunselected with respect to fertility-related features were assessed. TP53 polymorphisms and haplotypes were identified by\namplification refractory mutation system polymerase chain reaction. TP53 PIN3 and PEX4 were associated with both END\n(P ¼ 0.042 and P ¼ 0.007, respectively) and IVF ( P ¼ 0.004 and P ¼ 0.009, respectively) when compared with women both\nselected and unselected for fertility-related features. Haplotypes D-C and N-C were related to higher risk for END ( P ¼ 0.002,\nP ¼ 0.001, respectively) and failure of IVF ( P ¼ 0.018 and P ¼ 0.002, respectively) when compared with the Fertile group.\nThese results support that specific TP53 haplotypes are associated with an increased risk of END-associated infertility and\nwith post-IVF failure.\nCell Death and Disease (2012) 3, e392; doi:10.1038/cddis.2012.116; published online 27 September 2012\nSubject Category: Cancer\nTP53 encodes the multi-functional tumor suppressor tran-\nscription factor p53 which has a crucial role in maintaining\ngenomic stability in somatic cells exposed to oncogenic or\ngenotoxic stress, thus preventing tumor formation.\n1 In\nresponse to a wide range of stress signals, p53 accumulates\nin the nucleus and regulates the expression of a large panel of\ngenes involved in the control of cell cycle arrest, apoptosis,\ncell senescence, DNA repair and energy metabolism. One of\nthe transcriptional targets of p53 is leukemia inhibitory factor\n(LIF), the gene encoding LIF. LIF is a secreted cytokine with\nbroad roles in the control of lymphocyte proliferation and\ndifferentiation. It has also been identified as a critical factor for\nblastocyst implantation.\n2 Control of p53 over LIF expression is\noperated through a p53-response element located in intron 1\nand conserved in both mouse and human LIF genes.3\nRecent studies have demonstrated that p53 regulates\nfemale reproduction and blastocyst implantation through\nLIF. Implantation is a critical step in mammalian embryonic\ndevelopment during which the blastocyst establishes close\ninteractions with the uterus, leading to the formation of the\nplacenta supporting fetal development.\n4 Hu et al. 4 have\ndemonstrated that p53 regulates LIF expression in the uterus\nof female mice. p53-deficient mice express lower levels of LIF\nthan their p53-competent counterparts and show impaired\nblastocyst implantation and consequently, impaired fertility.\nThere is strong evidence that genes at critical regulatory\nnodes in the p53 pathway are under evolutionary selection\n5,6\nand that SNPs in the p53 pathway influence human fertility. 7\nOf these, one of the most studied is TP53 polymorphism in\nexon 4 (PEX4 of the TP53 gene), widely known as p.P72R\n(C/G, rs1042522). This single-nucleotide polymorphism\n(SNP) located at the second position of the codon 72 consist\nin either an ancestral C allele whose frequency in African\npopulations is around 0.70 or a derived G allele whose\nfrequency in European and Asian populations varies from\naround 0.50 to 0.80. Presence of the C allele results in a\nproline in codon 72, and presence of the G allele, in an\narginine. These polymorphic protein variants significantly\ndiffer in their biological properties and there is evidence that\nR72p53 has higher transcriptional activity toward a particular\nsubset of p53 target genes, including LIF, than P72p53.\n8\nPrevious studies have identified an association between\nTP53 PEX4 and infertility 7 or endometriosis (END). 9–11 It has\nbeen suggested that the effect of PEX4 on LIF expression\nand fertility may account for population differences in the\ndistribution of PEX4 alleles in different parts of the world.\n1Department of Genetics and Molecular Biology, Federal University of Rio Grande do Sul, Porto Alegre, Brazil; 2Department of Obstetrics and Gynecology, Federal\nUniversity of Rio Grande do Sul, Porto Alegre, Brazil;3Post-Graduate Program in Genetics and Molecular Biology, Federal University of Rio Grande do Sul, Porto Alegre,\nBrazil; 4International Prevention Research Institute, Lyon, France;5Medical Genetics Service, Hospital de Clı´nicas de Porto Alegre, and National Institute of Science and\nTechnology in Populational Medical Genetics (INAGEMP), Porto Alegre, Brazil and6Genomic Medicine Laboratory, Experimental Research Center, Hospital de Clı´nicas\nde Porto Alegre, Porto Alegre, Brazil\n*Corresponding author: P Ashton-Prolla, Departamento de Gene´tica, UFRGS e Servic¸o de Gene´tica Me´dica e Centro de Pesquisa Experimental, Hospital de Clı´nicas\nde Porto Alegre, Rua Ramiro Barcelos, 2350, Porto Alegre 90035-903, Brazil. Tel: +55 51 3359 8010; Fax: +55 51 3359 8760; E-mail: pprolla@hcpa.ufrgs. br\nReceived 30.4.12; revised 21.6.12; accepted 02.7.12; Edited by Y Shi\nKeywords: TP53; single-nucleotide polymorphisms; IVF; endometriosis\nAbbreviations: TP53, tumor protein p53; LIF, leukemia inhibitory factor; PIN2, polymorphism in intron 2; PIN3, polymorphism in intron 3; PEX4, polymorphism in\nexon 4; SNP, single-nucleotide polymorphism; END, endometriosis; IVF, in vitro fertilization; D, duplicated allele; N, non-duplicated allele\nCitation: Cell Death and Disease (2012) 3, e392; doi:10.1038/cddis.2012.116\n& 2012 Macmillan Publishers Limited All rights reserved 2041-4889/12\nwww.nature.com/cddis\n\nThese differences may reflect subtle adaptation to environ-\nmental constraints affecting fertility. However, the magnitude\nof the PEX4 effect on infertility associated with different\npathological causes remains controversial.\n12,13\nPEX4 is in strong linkage disequilibrium with another common\npolymorphism located in its close vicinity, PIN2 (polymorphism\nin intron 2; rs1642785; G/C). The PIN2 G allele has been\nassociated with human papillomavirus persistence 14 and\nindividuals with two copies of the PIN2 G allele have been\nreported as having an increased risk of osteosarcoma. 15\nRecently, it has been shown that another polymorphism\nin intron 3 of the TP53 gene, PIN3 (Polymorphism in\nIntron 3, rs17878362, 16 bp duplication, N ¼ non-duplicated,\nD ¼ duplicated) overlaps with a G-quadruplex motif, which regu-\nlates p53 mRNA splicing generating an alternatively spliced\nform, which supports the synthesis of an isoform of p53 lacking\nthe N-terminal transactivation domain (Delta40p53).\n16 PIN3 D\nallele is associated with increased risk of colorectal, 17 lung18\nand breast cancer, 19 whereas the N allele has been reported\nin association with an average acceleration of 19 years in the\nmean age at first cancer diagnosis in a Brazilian cohort ofTP53\ngermline mutation carriers.20 The effects of this polymorphism\nin END or infertility have not been investigated so far.\nAlthough the association between END and infertility is well\nknown (END affects up to 50% of women with infertility), 21 the\ncause of infertility in the disease is not fully understood but\nis thought to involve hormonal, 22 immunological,23 genetic,24\nproliferative (endometrial) and uterine alterations. 25 We\nhypothesized that TP53 polymorphisms that alter p53 function\nmay be associated with in vitro fertilization (IVF) failure and\nwith END-associated infertility.\nResults\nPatients and healthy study subjects did not differ significantly\nregarding self-attributed skin color (Supplementary Table S1).\nOverall, a self-denomination of ‘white’ color predominated in\nall study subgroups (END, FIV, Unselected and Fertile). In\nterms of reproductive history, the mean number of pregnan-\ncies in women of the fertile and unselected for fertility groups\nwas 3.62 ±1.9 and 3.22 ±2.1, respectively. In the later,\nnulliparity was observed in 2.6%.\nWomen in the fertile and unselected for fertility groups\npresented higher mean age at recruitment (42.68±12.8 years\nand 43.2 ±12.7 years, respectively) as compared with END\n(32.87±4.7 years) and IVF (31.65 ±3.2 years) groups.\nHardy–Weinberg equilibrium was achieved in all study groups\nfor PIN2, PIN3 and PEX4 (all P40.05, Supplemental\nMaterials, Table S2. Genotypic and allelic frequencies of the\nTP53 polymorphisms are shown in Table 1. In all four study\nsubgroups, PIN3 and PEX4 allele frequencies did not differ\nsignificantly from those previously described in European\npopulations (Supplementary Table S3).\nSingle marker analysis (Table 1) revealed a significant\nassociation between PIN2 (rs1642785) genotypes and IVF\n(P ¼ 0.016), and a borderline association with the END group\n(P ¼ 0.052) when compared with the Fertile group. There was\nan increased frequency of the PIN2 C allele in both the END\nand IVF groups. When analyzing TP53 PIN3 (rs17878362)\npolymorphism, a clear difference between IVF and END\ngroups was observed when compared with the Fertile group.\nAllele D (the duplicated allele) was enriched in patients in both\ngroups as compared with Fertile (P ¼ 0.042 and Po0.0004 for\nthe END and IVF groups, respectively). For TP53 PEX4\n(rs1042522), a statistically significant difference between both\nthe END and IVF groups and the Fertile group was also\ndemonstrated, with enrichment of the PEX4 C allele in both\ngroups (P ¼ 0.007 and P ¼ 0.009, respectively).\nWhen the Fertile and Unselected groups were compared,\nwe observed that the allelic frequencies of PIN2 G and PEX4\nG were significantly higher in the Fertile group, whereas PIN2\nand PEX4 genotype distribution did not differ between groups.\nTable 1 Genotypic and allelic frequencies of selected TP53 polymorphisms between Fertility Unselected, Fertile, END and IVF groups\nTP53 Unselected, n (%) Fertile, n (%) P-valuea END, n (%) P-valueb P-valuec IVF, n (%) P-valued P-valuee\nPIN2 GG 166 (55.3) 88 (65.7) 0.114 53 (54.1) 0.304 0.052 63 (54.8) 0.049 0.016\nrs1642785 GC 112 (37.3) 40 (29.9) 33 (33.7) 35 (30.4)\nCC 22 (7.3) 6 (4.5) 12 (12.2) 17 (14.8)\nG 444 (74.0) 216 (80.6) 0.007 139 (70.9) 0.397 0.015 161 (70.0) 0.245 0.005\nC 156 (26.0) 52 (19.4) 57 (29.1) 69 (30.0)\nPIN3 NN 222 (74.0) 94 (70.1) 0.658 49 (50.0) o0.001 0.042 72 (62.6) o0.001 0.004\nrs17878362 ND 70 (23.3) 35 (26.1) 32 (32.7) 29 (25.2)\nDD 8 (2.7) 5 (3.7) 17 (17.3) 14 (12.2)\nN 514 (85.7) 223 (83.2) 0.350 130 (66.3) o0.001 0.005 173 (75.2) o0.001 0.027\nD 86 (14.3) 45 (16.8) 66 (33.7) 57 (24.8)\nPEX4 GG 158 (52.7) 89 (66.4) 0.013 50 (51.0) 0.535 0.007 63 (54.8) 0.159 0.009\nrs1042522 GC 114 (38.0) 40 (29.9) 35 (35.7) 35 (30.4)\nCC 28 (9.3) 5 (3.7) 13 (13.3) 17 (14.8)\nG 430 (71.7) 218 (81.3) 0.002 135 (68.9) 0.455 0.001 161 (0.70) 0.635 0.003\nC 170 (28.3) 50 (18.7) 61 (31.1) 69 (0.30)\nAbbreviations: D, duplicated; END, endometriosis; IVF, in vitro fertilization; N, non-duplicated.\naw2-test, significant difference observed between women unselected for fertility and Fertile women.\nbw2-test, significant difference observed between END patients and women unselected for fertility.\ncw2-test, significant difference observed between END patients and Fertile group.\ndw2-test, significant difference observed between IVF patients and women unselected for fertility.\new2-test, significant difference observed between IVF patients and Fertile group.\nIVF group: women with recurrent failure of IVF; END group: infertile women with minimal or mild endometriosis; Fertile: Fertile women; Unselected: women unselected\nwith respect to fertility or infertility-related symptoms.\nTP53 polymorphisms in infertility\nDD Paskulin et al\n2\nCell Death and Disease\n\nSimilarly, PIN3 genotypic or allelic frequencies did not differ\nbetween groups (Table 1). For both the END and IVF groups, the\nallelic frequencies of PIN2, PIN3 and PEX4 differed significantly\nfrom those observed in the Fertile group. The allelic frequencies\nof PIN3 in infertile women (either END and IVF groups) differed\nsignificantly from both the Fertile and Unselected groups.\nHaplotype analysis showed strong linkage disequilibrium\nbetween TP53 PIN2 and PEX4 ( D0 ¼ 1; r2 ¼ 0.94 in all studies\ngroups, Supplementary Table S4) as previously described. 20\nTherefore, we have only considered TP53 PIN3 and TP53\nPEX4 in further analyses and in our discussion. We carried out\na binary logistic regression analysis to evaluate the effect of\nTP53 haplotypes with regard to END and IVF. Figure 1 shows\nthe distribution of the most frequent haplotypes encountered\n(see Figure 1). Table 2 shows the odds ratios for the END and\nIVF groups of the most frequent haplotypes when compared with\nthe reference N-G haplotype. Haplotypes D-C and N-C were\nrelated to higher risk for END (P ¼ 0.002,P ¼ 0.001, respectively)\nand failure of IVF (P ¼ 0.018 and P ¼ 0.002, respectively) when\ncompared with the Fertile group. However, when the Unselected\ngroup (unselected for fertility) was used as the comparison\ngroup in the logistic regression model, the risk association with\nhaplotypes D-C and N-C was not observed (data not shown).\nDiscussion\nIn this study, we have analyzed the distribution of three common\npolymorphisms in the TP53 gene (PIN2, PIN3 and PEX4) in\ninfertile women with failure of IVF treatment or with END-\nassociated infertility. Our results demonstrate an association\nbetween these two forms of infertility and TP53 alleles PIN3 D\nand PEX4 C, suggesting that variations in p53 activity\nspecified by these polymorphisms may be involved in the\npathogenesis of both conditions. These results support\npreviously reported observations on associations between\nPEX4 and infertility, in particular IVF failure. Furthermore,\nthese results provide clear evidence in favor of an association\nbetween TP53 polymorphism and infertility-related END.\nRegarding TP53 PIN3, several studies have evaluated the\nassociation between this polymorphism and lung 18 or breast\ncancer,19 but to our knowledge, no previous study has\nanalyzed its association with infertility or END.\nFigure 1 Distribution of the most frequent haplotypes among Unselected, Fertile, END and IVF groups. Haplotypes frequencies are shown as ‘%’. Haplotypes wer e\nconstructed as PIN2 (G/C) – PIN3 (N/D) – PEX4 (C/G)\nTable 2 Binary logistic regression model for TP53 haplotypes regarding PIN3\n(N/D) and PEX4 (C/G) using the Fertile group as reference\nEND IVF\nHaplotype P-value OR (95% CI) P-value OR (95% CI)\nN-G\nD-C 0.002 2.1 (1.3–3.5) 0.018 1.7 (1.1–2.7)\nN-C 0.001 4.9 (2.1–12.4) 0.002 4.1 (1.6–9.8)\nAbbreviations: CI, confidence interval; D, duplicated; END, endometriosis;\nIVF, in vitro fertilization; N, non-duplicated; OR, odds ratio.\nOR (95% CI) was calculated by binary logistic regression analysis. IVF group:\nwomen with recurrent failure of IVF; END group: infertile women with minimal or\nmild endometriosis.\nTP53 polymorphisms in infertility\nDD Paskulin et al\n3\nCell Death and Disease\n\nPrevious studies have shown associations between PEX4\nC allele and END, 10,26,27 whereas others fail to demonstrate\nthis association. 11,12 These controversies may be due to\nthe environmental and genetic background of the studied\npopulations but also because of differences in illness\nclassifications (END is sometimes asymptomatic and often\ncan only be diagnosed by laparoscopy). In our study, the END\ngroup was carefully diagnosed according to the American\nSociety for Reproductive Medicine (ASRM) and women with\nEND were excluded from both IVF and Fertile groups after\nlaparoscopic examination. In addition, all four study groups\ndescribed here were quite homogeneous in terms of self-\nreported skin color (a feature used as proxy for ‘race’ or\nancestry background in Brazil) corroborating with previous\npopulation-based studies that demonstrate predominance of\nEuropean genomes in this specific region.\n28–32 This observa-\ntion was further confirmed by comparative analysis of PIN3\nand PEX4 allele frequencies encountered here and those\npreviously described in European/European-derived and\nAfrican/African-derived populations, showing that in all four\nstudy groups, allelic distribution was not statistically different\nfrom the observed in Europeans/Europeans-derived.\nKay et al.\n33 were the first to associate TP53 PEX4 C allele\nwith women experiencing recurrent implantation failure. Other\nstudies also associated the PEX4 C allele with the occurrence\nof idiopathic recurrent miscarriages\n34 and implantation fail-\nure,35 and Kang et al. 7 demonstrated that PEX4 C was\nsignificantly enriched among IVF patients, serving as risk\nfactor for implantation failure. Our results are in agreement\nwith these previous findings regarding the TP53 PEX4 C allele\nand confirm this allele as a risk factor for both END-associated\ninfertility and IVF failure in a different sample set.\nThese results and the findings of our study suggest that\nPIN3 and PEX4 polymorphisms present specific functional\ndifferences in p53 protein variants, having an impact on\nevents that are critical for embryo implantation and/or early\ndevelopment. In the case of PEX4, there is experimental\nevidence from cell and animal studies that the p53 protein\nencoded by the PEX4 C allele (P72p53) is more efficient in\ninitiating senescence that the product of the PEX4 G allele\n(R72p53), which in turn appears to have a stronger effect on\np53-mediated apoptosis and suppression of cell transforma-\ntion.\n36 In the case of PIN3, presence of the TP53 PIN3 D allele\nhas been associated with reduced levels of TP53 mRNA in\nlymphoblastoid cell lines. 17 Whether this effect also occurs\nin vivo remains to be determined. Marcel et al. 16 demon-\nstrated that TP53 PIN3 is located within a GC-rich region of\nintron 3 that form G-Quaduplex structures, which modulate\nsplicing of intron 2. In silico models predict that PIN3 may alter\nthe topology of these G-quadruplex structures, thus modifying\nthe patterns of p53 mRNA isoform expression. The p53\nisoform encoded by alternatively spliced p53 retaining intron 2\nlacks the N-terminal domain containing the main transactiva-\ntion activity of p53, thus resulting in an N-terminally truncated\nprotein, which binds DNA but does not activate transcription\nthrough p53-response elements. It is important to emphasize\nthat in our study TP53 PIN3 presented an allelic distribution\nthat was significantly different in infertile women (either END\nor IVF groups) when compared with women selected for\nfertility but also when compared with women from a\ncommunity sample and unselected for fertility, suggesting\nthat genetic variations in PIN3 may have a critical effect on\ninfertility. Further experimental studies are needed to evaluate\nthe possible impact of p53 isoforms in regulating these\nbiological events, especially their impact on transactivation of\nkey genes involved in the early stages of gestation, such as\nLIF. Haplotypes D-C and N-C were related to higher risk for\nEND and IVF only when a group of women selected in favor of\nnormal fertility (the Fertile group) was used as comparison\ngroup; this was not observed when the comparison group\nincluded women unselected for reproductive history. This\nobservation suggests that specific haplotypes of TP53 may be\nassociated with high fertility features. Given the associations\nbetween specific SNPs and infertility, it is reasonable to\nassume that particular combinations of SNPs might provide a\ngenetic marker for women with high fertility features.\nOur results support that TP53 polymorphisms have a role in\nboth END-associated infertility and IVF failure; although\ncurrent evidence points to a strong effect of the PEX4\npolymorphism in embryo implantation and fertility, other SNPs\nin TP53, especially PIN3, may have a key role in the\nmodulation of this process and in other biological processes\nrelated to early embryonic development. PIN3 was the only\nSNP that showed differential frequencies in infertile women\n(either END and IVF groups) when compared with either\nfertility-selected or unselected groups, whereas PIN2 and\nPEX4 only showed a differential distribution in END and IVF\npatients when compared with a group of patients at the other\nextreme of the phenotype (fertile group).\nIn conclusion, the data presented here add to the current\nevidence that variations in expression and activity of p53 may\nhave an effect on the expression of key genes related to the\ncontrol of cellular growth and invasion, which have been\nassociated with END (BAX, FAS, PIG11, PTEN), as well as on\ngenes associated with embryo implantation ( LIF). Infertility\nassociated to END could be related, at least in part, to embryo\nimplantation failure in a mechanism similar to that seen in\nother infertile women without END. It may also involve other\nmechanisms affecting early embryonic development as well\nas cell–cell communications during the pre-implantation and\nimplantation phases. In agreement with this hypothesis,\nprevious studies have demonstrated lower implantation and\npregnancy rates in endometriotic patients.\n37 TP53 poly-\nmorphisms, especially PIN3 and PEX4 may have an interest\nas biomarkers and could add to the development of a clinically\nrelevant genetic profile that would be of great help for\nclinicians to identify patients at higher risk for IVF failure.\nThe results of this study should be confirmed in larger cohorts\nwith well defined phenotypes of END and infertility and long-\nterm follow-up data. They also emphasize the importance of a\nclear definition of clinical phenotypes and of study design when\nanalyzing the effects of specific polymorphisms on fertility.\nMaterials and Methods\nPatients and subjects. All patients and subjects were informed about the\nprocedures of the study when invited to participate and signed a consent form at\ninclusion. The research project was approved by the Institutional Ethics Committee\n(Hospital de Clinicas de Porto Alegre – GPPG 05-182; GPPG 09-430).\nAt inclusion, patients and subjects were also asked to provide a description of\ntheir perceived skin color. In Brazil, skin color is normally used to define an equivalent\nto ‘race’ or ancestry background.\n32,38 We used the words ‘White’ and ‘non-White’\nTP53 polymorphisms in infertility\nDD Paskulin et al\n4\nCell Death and Disease\n\nto identify women who defined themselves with some term that suggests only\nEuropean ancestry and with other terms that suggest some level of African ancestry\n(such as mulato or pardo), respectively. No term that reports some level of\nAmerindian ancestry was used by volunteers.\nPatients and subjects were divided into four study groups. The IVF Group\nconsisted of 115 women ( o35 years) with at least one IVF failure, defined as a\nfailure after IVF cycle treatment with transfer of two or more top quality embryos\n(8 cell embryos with o20% fragmentation). Briefly, inclusion criteria of this group\nwere: age o35 years, exclusion of END by laparoscopy and the main factor was\nof mild masculine (oligospermia) or tubal origin. All patients in this group were\nsubmitted to conventional IVF. Patients with previous thyroid disease, positive anti-\nlupus or anti-cardiolipin antibodies and trombophilias were also excluded from our\nsample. Controlled ovarian hyperstimulation was performed with the use of\nrecombinant human FSH and pituitary suppression with GnRh antagonist (fixed\nday-6 protocol). Ovulation was induced by 6500 IU recombinant hCG when at least\nthree follicles had reached a diameter of 417 mm, and transvaginal follicle\naspiration was performed 36 h later under ultrasound guidance. Embryos were\nclassified according to the cumulative embryo classification, taking into account\ncleavage speed, blastomere symmetry, extent of fragmentation and the presence or\nabsence of multinucleated blastomeres.\nThe END group comprised 98 infertile women with minimal or mild END as\ndiagnosed by laparoscopy recruited at the Gynecology Service of Hospital de\nClinicas de Porto Alegre (HCPA), in Southern Brazil. Infertility was defined as the\ninability of a couple to achieve pregnancy after 1 year of regular unprotected sexual\nintercourse.\n39 Other causes of infertility were excluded by hysterosalpingography,\nsperm evaluation and hormonal measurements whenever necessary. END\ndiagnosed during laparoscopy was categorized according to the classification\nproposed by the ASRM.\n39\nThe Fertile group consisted of 134 women with no history of infertility, who\nalready had children without any difficulties or assisted reproduction and underwent\nlaparoscopy for tubal ligation at HCPA. END was excluded in women from IVF and\nFertile groups. In addition, we studied a group of 300 asymptomatic women, who\nvolunteered for a community-based breast cancer screening program in Southern\nBrazil (from the same geographic recruitment area of the patients included in the\nIVF and Fertile groups). This group (‘Unselected’) was unselected with respect to\nfertility or infertility-related symptoms, as described elsewhere.\n40\nGenotyping. Genomic DNA was extracted from peripheral blood using the\nIlustra blood genomic Prep Mini spin Kit (GE Healthcare, Piscataway, NJ, USA) as\ndescribed by the manufacturer. Genotypes and haplotypes defined by the three\nTP53 gene polymorphisms (PIN2 rs1642785 G/C, PIN3 rs17878362 16 pb\nduplication and PEX4 rs1042522 C/G) were determined by Amplification\nRefractory Mutation System as previously described.\n20\nStatistical analysis. The clinical characteristics of the women in all study\ngroups were compared by one-way analysis of variance. Differences in genotype/\nallele distribution between IVF, END, Fertile and Unselected groups were\nevaluated using w\n2-analysis, also used to test for Hardy–Weinberg equilibrium.\nLinkage disequilibrium was assessed calculating D0 value (the relative magnitude\nof D as compared with its theoretical maximum, calculated as D/Dmax)a s\ndescribed by Lewontin. 41\nBinary logistic regression analysis was carried out to estimate the odds ratios with\n95% confidence intervals in order to assess the influence of TP53 haplotypes for\nEND and IVF using the Fertile group as reference. Haplotype frequencies were\ncalculated by direct count. Statistical analysis was performed using the SPSS 18.0\nstatistical package. All reported P-values are two-tailed and considered statistically\nsignificant when 0.05X.\nConflict of Interest\nThe authors declare no conflict of interest.\nAcknowledgements. The work of DDP was supported by fellowships from\nCAPES and CNPQ (Brazil). The study was supported in part by grants\nfrom GlaxoSmithKline Oncology (Ethnic Research Initiative Grant Award\n2009), UK; CNPq to PA-P (Grant 307779 2009-2), Brazil; FAPERGS-PPSUS\n(Grant no. 09/0103-0), FAPERGS PRONEX (Grant no. 10/0051-9) and Fundo\nde Incentivo a Pesquisa e Eventos, Hospital de Clı´nicas de Porto Alegre (GPPG no.\n09430), Brazil.\n1. Levine AJ, Hu W, Feng Z. The P53 pathway: what questions remain to be explored? Cell\nDeath Differ 2006; 13: 1027–1036.\n2. Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F et al. 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To view a copy of this license,\nvisit http://creativecommons.org/licenses/by-nc-nd/3.0/\nSupplementary Information accompanies the paper on Cell Death and Disease website (http://www.nature.com/cddis)\nTP53 polymorphisms in infertility\nDD Paskulin et al\n6\nCell Death and Disease","source_license":"CC0","license_restricted":false}