Original Paper
HOR MON E RE SE ARCH I N PÆDIATRIC S
Received: June 26, 2014 Accepted: July 21, 2014 Published online: September 19, 2014
Horm Res Paediatr 2014;82:303–309 DOI: 10.1159/000366079
Mitochondrial DNA in Placenta: Associations with Fetal Growth and Superoxide Dismutase Activity Marta Díaz a, b Gemma Aragonés c David Sánchez-Infantes a, b Judit Bassols d Míriam Pérez-Cruz a Francis de Zegher e Abel Lopez-Bermejo d Lourdes Ibáñez a, b
a
Hospital Sant Joan de Déu, University of Barcelona, Esplugues, b Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Madrid, c Department of Medicine and Surgery, Universitat Rovira i Virgili (URV), Tarragona, and d Department of Pediatrics, Dr. Josep Trueta Hospital, and Girona Institute for Biomedical Research, Girona, Spain; e Department of Development and Regeneration, University of Leuven, Leuven, Belgium
Abstract Background: Prenatal growth restraint is associated with increased oxidative stress – as judged by mitochondrial dysfunction – in pregnancies complicated by preeclampsia or diabetes, but it is uncertain whether this is also the case in uncomplicated pregnancies. We assessed the link between fetal growth restraint and placental mitochondrial dysfunction, as reflected by changes in mitochondrial DNA (mtDNA) content and superoxide dismutase (SOD) activity. Methods: After uncomplicated pregnancies, placentas (n = 48) were collected at term delivery of singleton infants who were appropriate for gestational age (AGA) or small for gestational age (SGA) (n = 24 in each subgroup). Placental mtDNA content was assessed by real-time PCR, placental SOD activity by colorimetry, and citrate synthase activity – to determine mitochondrial mass – by the spectrophotometric method. Results: Placentas of SGA infants had a lower mtDNA content (p = 0.015) and a higher SOD activity (p = 0.001) than
© 2014 S. Karger AG, Basel 1663–2818/14/0825–0303$39.50/0 E-Mail [email protected] www.karger.com/hrp
those of AGA controls. These differences were maintained after normalization of the mtDNA content by citrate synthase activity. In placentas of SGA infants, there was a negative association between mtDNA content and SOD activity (r = –0.58, p = 0.008). Conclusions: Fetal growth restraint is accompanied by adaptive changes in the mitochondrial function of the placenta, also in uncomplicated pregnancies. © 2014 S. Karger AG, Basel
Introduction
Oxidative stress and mitochondrial dysfunction underlie the development of many pathological conditions, including type 2 diabetes and cardiovascular disease [1– 3]. Fetal and early postnatal protein malnutrition may also result in long-term mitochondrial abnormalities that contribute to the development of the metabolic syndrome [4, 5]. In turn, oxidative stress results from accumulation of toxic reactive oxygen species (ROS) generated by mitochondria due to an imbalance between pro-oxidant production and antioxidant defense [6].
Lourdes Ibáñez, MD, PhD Endocrinology Unit, Hospital Sant Joan de Déu, University of Barcelona Passeig de Sant Joan de Déu, 2 ES–08950 Esplugues/Barcelona (Spain) E-Mail libanez @ hsjdbcn.org
Downloaded by: UCSF Library & CKM 169.230.243.252 - 3/11/2015 10:22:26 AM
Key Words Placenta · Fetal growth · Mitochondrial DNA · Superoxide dismutase activity · Small for gestational age
Subjects and Methods Study Population The study cohort consisted of 48 mother-placenta-infant trios recruited at the Hospital Sant Joan de Déu, Barcelona, between May 2006 and September 2009 (see flowchart in fig. 1). Inclusion criteria were: (1) singleton pregnancy followed from the first trimester at the Hospital Sant Joan de Déu, Barcelona; ab-
304
Horm Res Paediatr 2014;82:303–309 DOI: 10.1159/000366079
sence of maternal pathology (hypertension, gestational diabetes, preeclampsia); absence of maternal smoking and alcoholism. (2) Placenta collected for research purposes at delivery, after informed written consent by the pregnant mother. (3) Infants born at term (37–42 weeks); birth weight range between –1.1 and +1.1 standard deviation (SD, AGA) and below –2 SD (SGA). (4) Co-availability of maternal, placental and neonatal information (logistic restraints, particularly for nighttime collection of placentas). (5) Written, informed consent, obtained in the third trimester. Exclusion criteria were congenital malformations or other complications at birth (need for resuscitation or parenteral nutrition). Information on maternal age at conception, parity, height, as well as on body weight and body mass index before gestation and also before delivery was retrieved from the mothers’ clinical records, along with placental weight. Gestational age was calculated by last menses and confirmed by first-trimester ultrasound (∼10 weeks). The weight and length of the newborns were measured at birth. All SGA infants were classified as having type II, asymmetric growth restriction. Placenta Collection and Tissue Homogenate for Assessing SOD Activity Placentas were collected and weighed immediately after delivery, and placental tissue was then obtained from the maternal side, as described [22]. Placental SD scores (SDS) were derived from published gender- and gestational-age-specific centile curves for placental weight [23]. Samples were frozen in liquid nitrogen and stored at –80 ° C until DNA extraction. 100 mg of frozen placental tissue was rinsed with phosphate-buffered saline, pH 7.4, to remove any red blood cells and clots. Subsequently, the tissue was homogenized in 5 ml of cold 20 mM Hepes buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose, and centrifuged at 1,500 g for 5 min at a temperature of 4 ° C; the supernatant was used for the SOD assay. The protein concentration of the supernatant was determined by nanodrop. SOD activity (both cytosolic and mitochondrial) was assessed using a commercial ELISA kit, according to the manufacturer’s protocol (Cayman Chemical Co., Ann Arbor, Mich., USA) [24]. This method is based on the use of a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Results were expressed as units of SOD per milligram of protein (U/ mg). The intra- and interassay coefficients of variation were 3.2 and 3.7%, respectively.
Assessment of Citrate Synthase Activity and Quantification of Mitochondrial Mass To determine citrate synthase (CS) activity, a new protein homogenate was obtained using the CellLytic MT (Sigma-Aldrich, St. Louis, Mo., USA). Briefly, 100 mg of frozen tissue was homogenized in 0.5 ml of buffer and centrifuged for 10 min at 12,000 g to pellet the tissue debris. The supernatant was used for protein determination (nanodrop); 1 μg of protein was used for the assessment of CS activity (Citrate Synthase Assay Kit; Sigma-Aldrich). To determine the mitochondrial mass, the ratio [mtDNA/nuclear (n)DNA]/CS was calculated. This ratio is representative of the number of mtDNA copies within the mitochondrial mass [25].
Díaz et al.
Downloaded by: UCSF Library & CKM 169.230.243.252 - 3/11/2015 10:22:26 AM
Mitochondria are the major source of ROS because of their key role in ATP generation [6]. Superoxide dismutase (SOD), catalase and glutathione peroxidase are the main enzymes involved in the preservation of redox homeostasis, SOD being essential for the survival of aerobic organisms [7]. Under physiological conditions, the antioxidant defense systems repress oxidative stress; however, under pathological conditions, overproduction of ROS initiates oxidative reactions via nuclear factor-κB activation, leading to oxidative damage within the mitochondria and in cellular proteins, lipids and nucleic acids [8]. The activation of this metabolic pathway also induces the expression of proinflammatory molecules [9] and causes mutations and quantitative changes in mitochondrial DNA (mtDNA) inducing sublethal mitochondrial damage and thereby generating an inflammatory vicious circle [10]. The extent of oxidative stress is usually estimated by quantifying the mtDNA copy number in lymphocytes and/or tissues and by measuring antioxidant enzyme activities [11]. For example, a decreased mtDNA content in peripheral leukocytes has been associated with type 2 diabetes in adults [12] and with insulin resistance in adolescents [13]. Prenatal growth restraint has been associated with increased oxidative stress in pregnancies complicated by preeclampsia or diabetes [14–16]. However, data on the link between prenatal growth restriction and placental mtDNA copy number in uncomplicated, term pregnancies resulting in the delivery of small-for-gestational-age (SGA) infants are scarce and controversial, mainly because the studied cohorts were heterogeneous and the assessments were performed at different timings (during or after delivery) and in different conditions (either in cord blood samples or in placental tissue). In addition, none of the available reports have simultaneously assessed the mtDNA content and enzymatic activities [14, 17–21]. Here, we compared the mtDNA content in placentas of appropriate-for-gestational-age (AGA) and SGA infants and studied whether the mtDNA content is associated with SOD antioxidant system activity.
Mother-newborn pairs (May 2006 to Sept. 2009) n = 10,281
Placenta collected for research purposes (logistic and ethical restraints)
n = 315 (155 girls, 160 boys)
Inclusion and exclusion criteria (see Subjects and Methods)
n = 133 (65 girls, 68 boys)
Available clinical and placental tissue for SOD activity and quantitative PCR
Fig. 1. Recruitment of the study popula-
AGA n = 24 (14 girls, 10 boys)
SGA n = 24 (11 girls, 13 boys)
tion.
Quantification of mtDNA Content An additional amount of 100 mg of frozen placenta was homogenized using a Polytron benchtop homogenizer (Kinematica AG, Littau-Luzern, Switzerland); total DNA (genomic and mitochondrial) was purified using commercial reagents (Promega, Madison, Wisc., USA). The mtDNA/nDNA ratio was determined by real-time quantitative PCR using the ABI 7500 thermocycler (Applied Biosystems, Foster City, Calif., USA). Cytochrome c oxidase II (CoxII) and β-actin are ubiquitous in the mitochondrial and nuclear genomes, respectively. Therefore, the mtDNA content was calculated as the amplification (Ct) of CoxII relative to the amplification (Ct) of β-actin to normalize for nDNA, the amount of which remains invariable in every single cell [26]. The CoxII region was amplified using the forward primer 5′-CCCCACATTAGGCTTAAAAACAGAT-3′ and the reverse primer 5′-TATACCCCCGGTCGTGTAGC-3′. The primers for the amplification of β-actin were: forward, 5′-AGAAAATCTGGCACCACACC-3′, and reverse, 5′-AACGGCAGAAGAGAGAACCA-3′. The primers were designed to avoid amplification of pseudogenes using the basic local alignment search tool (BLAST) of the National Center for Biotechnology Information [21]. Total DNA (3 ng) was amplified with the SYBR green method adding 200 nM of each primer to a final volume of 25 μl. The following quantification cycling protocol was
used: 95 ° C for 10 min for polymerase activation and 40 cycles at 95 ° C for 30 s and at 59 ° C for 1 min.
Placental mtDNA and Fetal Growth
Horm Res Paediatr 2014;82:303–309 DOI: 10.1159/000366079
305
Downloaded by: UCSF Library & CKM 169.230.243.252 - 3/11/2015 10:22:26 AM
Statistics and Ethics Statistical analyses were performed using SPSS 12.0 (SPSS, Inc., Chicago, Ill., USA). Results are expressed as the mean ± SEM. Nonparametric variables were mathematically transformed to improve symmetry. An unpaired t test was used to study differences in continuous variables among groups. The relation between variables was analyzed by simple correlation followed by multiple regression in a stepwise manner. The significance level was set at p < 0.05. Assuming α and β risks of 0.05 and 0.20, respectively, the study was powered to detect, in bilateral tests, differences in mtDNA content or in SOD activity of at least 0.8 SDS. These differences are relevant from a clinical point of view. The study was also powered to detect significant but modest correlations between these two variables (those with a Spearman correlation coefficient of at least 0.55). The study was approved by the Institutional Review Board of the Barcelona University Hospital of Sant Joan de Déu; written informed consent for placental collection was obtained before delivery (see inclusion criteria).
Table 1. Maternal, neonatal and placental data according to birth weight subgroup
Mothers Age, years Weight, kg Body mass index Pregnancy weight gain, kg Primiparous, % Vaginal delivery, % Newborns Gestational age, weeks Girls, % Birth weight, kg Birth weight SDS Birth length, cm Birth length SDS Placental weight, kg Placental weight SDS Placentas mtDNA content, copies/nDNA CS activity, μmol/min/g protein mtDNA/CS activity SOD activity, U/mg protein
AGA (n = 24)
SGA (n = 24)
p value
28.8 ± 1.2 62.2 ± 2.0 23.9 ± 0.9 14.7 ± 1.2 71 75
28.5 ± 1.1 56.4 ± 1.8 21.5 ± 0.6 11.2 ± 0.8 75 58
n.s. 0.035 0.041 0.018 n.s. n.s.
39.7 ± 0.2 58 3.19 ± 0.06 – 0.2 ± 0.1 50.1 ± 0.5 0.2 ± 0.3 0.66 ± 0.02 – 0.1 ± 0.1
38.1 ± 0.2 45 2.17 ± 0.06 – 2.4 ± 0.1 45.0 ± 0.4 – 1.9 ± 0.2 0.48 ± 0.01 – 1.3 ± 0.1
HOR MON E RE SE ARCH I N PÆDIATRIC S
Received: June 26, 2014 Accepted: July 21, 2014 Published online: September 19, 2014
Horm Res Paediatr 2014;82:303–309 DOI: 10.1159/000366079
Mitochondrial DNA in Placenta: Associations with Fetal Growth and Superoxide Dismutase Activity Marta Díaz a, b Gemma Aragonés c David Sánchez-Infantes a, b Judit Bassols d Míriam Pérez-Cruz a Francis de Zegher e Abel Lopez-Bermejo d Lourdes Ibáñez a, b
a
Hospital Sant Joan de Déu, University of Barcelona, Esplugues, b Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Madrid, c Department of Medicine and Surgery, Universitat Rovira i Virgili (URV), Tarragona, and d Department of Pediatrics, Dr. Josep Trueta Hospital, and Girona Institute for Biomedical Research, Girona, Spain; e Department of Development and Regeneration, University of Leuven, Leuven, Belgium
Abstract Background: Prenatal growth restraint is associated with increased oxidative stress – as judged by mitochondrial dysfunction – in pregnancies complicated by preeclampsia or diabetes, but it is uncertain whether this is also the case in uncomplicated pregnancies. We assessed the link between fetal growth restraint and placental mitochondrial dysfunction, as reflected by changes in mitochondrial DNA (mtDNA) content and superoxide dismutase (SOD) activity. Methods: After uncomplicated pregnancies, placentas (n = 48) were collected at term delivery of singleton infants who were appropriate for gestational age (AGA) or small for gestational age (SGA) (n = 24 in each subgroup). Placental mtDNA content was assessed by real-time PCR, placental SOD activity by colorimetry, and citrate synthase activity – to determine mitochondrial mass – by the spectrophotometric method. Results: Placentas of SGA infants had a lower mtDNA content (p = 0.015) and a higher SOD activity (p = 0.001) than
© 2014 S. Karger AG, Basel 1663–2818/14/0825–0303$39.50/0 E-Mail [email protected] www.karger.com/hrp
those of AGA controls. These differences were maintained after normalization of the mtDNA content by citrate synthase activity. In placentas of SGA infants, there was a negative association between mtDNA content and SOD activity (r = –0.58, p = 0.008). Conclusions: Fetal growth restraint is accompanied by adaptive changes in the mitochondrial function of the placenta, also in uncomplicated pregnancies. © 2014 S. Karger AG, Basel
Introduction
Oxidative stress and mitochondrial dysfunction underlie the development of many pathological conditions, including type 2 diabetes and cardiovascular disease [1– 3]. Fetal and early postnatal protein malnutrition may also result in long-term mitochondrial abnormalities that contribute to the development of the metabolic syndrome [4, 5]. In turn, oxidative stress results from accumulation of toxic reactive oxygen species (ROS) generated by mitochondria due to an imbalance between pro-oxidant production and antioxidant defense [6].
Lourdes Ibáñez, MD, PhD Endocrinology Unit, Hospital Sant Joan de Déu, University of Barcelona Passeig de Sant Joan de Déu, 2 ES–08950 Esplugues/Barcelona (Spain) E-Mail libanez @ hsjdbcn.org
Downloaded by: UCSF Library & CKM 169.230.243.252 - 3/11/2015 10:22:26 AM
Key Words Placenta · Fetal growth · Mitochondrial DNA · Superoxide dismutase activity · Small for gestational age
Subjects and Methods Study Population The study cohort consisted of 48 mother-placenta-infant trios recruited at the Hospital Sant Joan de Déu, Barcelona, between May 2006 and September 2009 (see flowchart in fig. 1). Inclusion criteria were: (1) singleton pregnancy followed from the first trimester at the Hospital Sant Joan de Déu, Barcelona; ab-
304
Horm Res Paediatr 2014;82:303–309 DOI: 10.1159/000366079
sence of maternal pathology (hypertension, gestational diabetes, preeclampsia); absence of maternal smoking and alcoholism. (2) Placenta collected for research purposes at delivery, after informed written consent by the pregnant mother. (3) Infants born at term (37–42 weeks); birth weight range between –1.1 and +1.1 standard deviation (SD, AGA) and below –2 SD (SGA). (4) Co-availability of maternal, placental and neonatal information (logistic restraints, particularly for nighttime collection of placentas). (5) Written, informed consent, obtained in the third trimester. Exclusion criteria were congenital malformations or other complications at birth (need for resuscitation or parenteral nutrition). Information on maternal age at conception, parity, height, as well as on body weight and body mass index before gestation and also before delivery was retrieved from the mothers’ clinical records, along with placental weight. Gestational age was calculated by last menses and confirmed by first-trimester ultrasound (∼10 weeks). The weight and length of the newborns were measured at birth. All SGA infants were classified as having type II, asymmetric growth restriction. Placenta Collection and Tissue Homogenate for Assessing SOD Activity Placentas were collected and weighed immediately after delivery, and placental tissue was then obtained from the maternal side, as described [22]. Placental SD scores (SDS) were derived from published gender- and gestational-age-specific centile curves for placental weight [23]. Samples were frozen in liquid nitrogen and stored at –80 ° C until DNA extraction. 100 mg of frozen placental tissue was rinsed with phosphate-buffered saline, pH 7.4, to remove any red blood cells and clots. Subsequently, the tissue was homogenized in 5 ml of cold 20 mM Hepes buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose, and centrifuged at 1,500 g for 5 min at a temperature of 4 ° C; the supernatant was used for the SOD assay. The protein concentration of the supernatant was determined by nanodrop. SOD activity (both cytosolic and mitochondrial) was assessed using a commercial ELISA kit, according to the manufacturer’s protocol (Cayman Chemical Co., Ann Arbor, Mich., USA) [24]. This method is based on the use of a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Results were expressed as units of SOD per milligram of protein (U/ mg). The intra- and interassay coefficients of variation were 3.2 and 3.7%, respectively.
Assessment of Citrate Synthase Activity and Quantification of Mitochondrial Mass To determine citrate synthase (CS) activity, a new protein homogenate was obtained using the CellLytic MT (Sigma-Aldrich, St. Louis, Mo., USA). Briefly, 100 mg of frozen tissue was homogenized in 0.5 ml of buffer and centrifuged for 10 min at 12,000 g to pellet the tissue debris. The supernatant was used for protein determination (nanodrop); 1 μg of protein was used for the assessment of CS activity (Citrate Synthase Assay Kit; Sigma-Aldrich). To determine the mitochondrial mass, the ratio [mtDNA/nuclear (n)DNA]/CS was calculated. This ratio is representative of the number of mtDNA copies within the mitochondrial mass [25].
Díaz et al.
Downloaded by: UCSF Library & CKM 169.230.243.252 - 3/11/2015 10:22:26 AM
Mitochondria are the major source of ROS because of their key role in ATP generation [6]. Superoxide dismutase (SOD), catalase and glutathione peroxidase are the main enzymes involved in the preservation of redox homeostasis, SOD being essential for the survival of aerobic organisms [7]. Under physiological conditions, the antioxidant defense systems repress oxidative stress; however, under pathological conditions, overproduction of ROS initiates oxidative reactions via nuclear factor-κB activation, leading to oxidative damage within the mitochondria and in cellular proteins, lipids and nucleic acids [8]. The activation of this metabolic pathway also induces the expression of proinflammatory molecules [9] and causes mutations and quantitative changes in mitochondrial DNA (mtDNA) inducing sublethal mitochondrial damage and thereby generating an inflammatory vicious circle [10]. The extent of oxidative stress is usually estimated by quantifying the mtDNA copy number in lymphocytes and/or tissues and by measuring antioxidant enzyme activities [11]. For example, a decreased mtDNA content in peripheral leukocytes has been associated with type 2 diabetes in adults [12] and with insulin resistance in adolescents [13]. Prenatal growth restraint has been associated with increased oxidative stress in pregnancies complicated by preeclampsia or diabetes [14–16]. However, data on the link between prenatal growth restriction and placental mtDNA copy number in uncomplicated, term pregnancies resulting in the delivery of small-for-gestational-age (SGA) infants are scarce and controversial, mainly because the studied cohorts were heterogeneous and the assessments were performed at different timings (during or after delivery) and in different conditions (either in cord blood samples or in placental tissue). In addition, none of the available reports have simultaneously assessed the mtDNA content and enzymatic activities [14, 17–21]. Here, we compared the mtDNA content in placentas of appropriate-for-gestational-age (AGA) and SGA infants and studied whether the mtDNA content is associated with SOD antioxidant system activity.
Mother-newborn pairs (May 2006 to Sept. 2009) n = 10,281
Placenta collected for research purposes (logistic and ethical restraints)
n = 315 (155 girls, 160 boys)
Inclusion and exclusion criteria (see Subjects and Methods)
n = 133 (65 girls, 68 boys)
Available clinical and placental tissue for SOD activity and quantitative PCR
Fig. 1. Recruitment of the study popula-
AGA n = 24 (14 girls, 10 boys)
SGA n = 24 (11 girls, 13 boys)
tion.
Quantification of mtDNA Content An additional amount of 100 mg of frozen placenta was homogenized using a Polytron benchtop homogenizer (Kinematica AG, Littau-Luzern, Switzerland); total DNA (genomic and mitochondrial) was purified using commercial reagents (Promega, Madison, Wisc., USA). The mtDNA/nDNA ratio was determined by real-time quantitative PCR using the ABI 7500 thermocycler (Applied Biosystems, Foster City, Calif., USA). Cytochrome c oxidase II (CoxII) and β-actin are ubiquitous in the mitochondrial and nuclear genomes, respectively. Therefore, the mtDNA content was calculated as the amplification (Ct) of CoxII relative to the amplification (Ct) of β-actin to normalize for nDNA, the amount of which remains invariable in every single cell [26]. The CoxII region was amplified using the forward primer 5′-CCCCACATTAGGCTTAAAAACAGAT-3′ and the reverse primer 5′-TATACCCCCGGTCGTGTAGC-3′. The primers for the amplification of β-actin were: forward, 5′-AGAAAATCTGGCACCACACC-3′, and reverse, 5′-AACGGCAGAAGAGAGAACCA-3′. The primers were designed to avoid amplification of pseudogenes using the basic local alignment search tool (BLAST) of the National Center for Biotechnology Information [21]. Total DNA (3 ng) was amplified with the SYBR green method adding 200 nM of each primer to a final volume of 25 μl. The following quantification cycling protocol was
used: 95 ° C for 10 min for polymerase activation and 40 cycles at 95 ° C for 30 s and at 59 ° C for 1 min.
Placental mtDNA and Fetal Growth
Horm Res Paediatr 2014;82:303–309 DOI: 10.1159/000366079
305
Downloaded by: UCSF Library & CKM 169.230.243.252 - 3/11/2015 10:22:26 AM
Statistics and Ethics Statistical analyses were performed using SPSS 12.0 (SPSS, Inc., Chicago, Ill., USA). Results are expressed as the mean ± SEM. Nonparametric variables were mathematically transformed to improve symmetry. An unpaired t test was used to study differences in continuous variables among groups. The relation between variables was analyzed by simple correlation followed by multiple regression in a stepwise manner. The significance level was set at p < 0.05. Assuming α and β risks of 0.05 and 0.20, respectively, the study was powered to detect, in bilateral tests, differences in mtDNA content or in SOD activity of at least 0.8 SDS. These differences are relevant from a clinical point of view. The study was also powered to detect significant but modest correlations between these two variables (those with a Spearman correlation coefficient of at least 0.55). The study was approved by the Institutional Review Board of the Barcelona University Hospital of Sant Joan de Déu; written informed consent for placental collection was obtained before delivery (see inclusion criteria).
Table 1. Maternal, neonatal and placental data according to birth weight subgroup
Mothers Age, years Weight, kg Body mass index Pregnancy weight gain, kg Primiparous, % Vaginal delivery, % Newborns Gestational age, weeks Girls, % Birth weight, kg Birth weight SDS Birth length, cm Birth length SDS Placental weight, kg Placental weight SDS Placentas mtDNA content, copies/nDNA CS activity, μmol/min/g protein mtDNA/CS activity SOD activity, U/mg protein
AGA (n = 24)
SGA (n = 24)
p value
28.8 ± 1.2 62.2 ± 2.0 23.9 ± 0.9 14.7 ± 1.2 71 75
28.5 ± 1.1 56.4 ± 1.8 21.5 ± 0.6 11.2 ± 0.8 75 58
n.s. 0.035 0.041 0.018 n.s. n.s.
39.7 ± 0.2 58 3.19 ± 0.06 – 0.2 ± 0.1 50.1 ± 0.5 0.2 ± 0.3 0.66 ± 0.02 – 0.1 ± 0.1
38.1 ± 0.2 45 2.17 ± 0.06 – 2.4 ± 0.1 45.0 ± 0.4 – 1.9 ± 0.2 0.48 ± 0.01 – 1.3 ± 0.1
