Development of an experimental model of brain tissue heterotopia in the lung

The presence of heterotopic brain tissue in the lung is a rare abnormality. The cases reported thus far are usually associated with neural tube defects (NTD). As there are no reports of experimental models of NTD that present this abnormality, the objective of the present study was to develop a surgical method of brain tissue heterotopia in the lung. We used 24 pregnant Swiss mice divided into two groups of 12 animals each, denoted 17GD and 18GD according to the gestational day (GD) when caesarean section was performed to collect the fetuses. Surgery was performed on the 15th GD, one fetus was removed by hysterectomy and its brain tissue was cut into small fragments and implanted in the lung of its litter mates. Thirty-four live fetuses were obtained from the 17GD group. Of these, eight (23.5%) were used as control (C), eight (23.5%) were sham operated (S) and 18 (52.9%) were used for pulmonary brain tissue implantation (PBI). Thirty live fetuses were obtained from the females of the 18GD group. Of these, eight (26.6%) were C, eight (26.6%) S and 14 (46.6%) were used for PBI. Histological examination of the fetal trunks showed implantation of GFAP-positive brain tissue in 85% of the fetuses of the 17GD group and in 100% of those of the 18GD group, with no significant difference between groups for any of the parameters analysed. The experimental model proved to be efficient and of relatively simple execution, showing complete integration of the brain tissue with pulmonary and pleural tissue and thus representing a model that will permit the study of different aspects of cell implantation and interaction.     

Assessment of AFP in amniotic fluid: comparison of three automated techniques

Ultrasound scanning is useful to detect neural tube defect (NTD) but scarcely distinguished between closed NTD and open NTD, which had very different prognosis. An amniotic fluid punction is thus mandatory to search for an increase in alpha foeto protein (AFP) levels and for the presence of acetylcholinesterase which identified open NTD. However, AFP levels fluctuate both with the gestational age and the assay used. Our aim was to establish normative values for AFP in amniotic fluid in the second half of pregnancy using three different immunoassays and to improve their clinical relevance. Amniotic fluid punctions were performed on 527 patients from 9 week of gestation (WG) to 37 WG either for maternal age, Trisomy 21 screening, increase in nucal translucency (control group, n = 527) or for suspicion of neural tube defect or abdominal defect (n = 5). AFP was measured using the immunoassay developed for serum AFP on the Access 2 system, the Immulite 2000 and the Advia Centaur. Results were expressed in ng/ml, multiple of the median (MoM) and percentiles. AFP decrease by 1.5 fold between 9 and 19 WG. When NTD was suspected, an increase in anmniotic AFP was observed (from 2.5 MoM to 9.3 MoM) confirming an open NTD. In conclusion, the assay developed on those 3 automates is suitable for the measurement of AFP in amniotic fluid.     

全反式维甲酸诱导骨骼畸形大鼠血清及羊水内碱性成纤维细胞生长因子的表达简

目的探究应用全反式维甲酸诱导骨骼畸形大鼠血清及羊水内碱性成纤维细胞生长因子（FGF2）的表达。方法选择孕10d的Wistar大鼠50只,体质量250～300g。分实验组和对照组.每组各25只。实验组予溶有全反式维甲酸的大豆油（40mg／mL）,按照135mg/hg以灌胃方式给药制作骨骼畸形胎鼠模型;对照组给予等体积的大豆油。孕20d时处死母鼠,采集母鼠血液,实验组每窝选择骨骼畸形胎鼠4只,对照组每窝随机选取胎鼠4只,抽取胎鼠羊水标本,应用酶联免疫吸附分析测定血液及羊水内FGF2浓度：利用游标卡尺测量胎鼠头臀长,双前肢各段及双后肢的长度。结果实验组胎鼠出现四肢发育不良、脊柱裂、下颌裂等畸形。实验组胎鼠头臀长、双前肢各段及双后肢长度与对照组相比.差异具有统计学意义（P〈0.05）。实验组母鼠血液及胎鼠羊水内FGF2的浓度与对照组相比[（24.124±1.271）pg／mL vs（27.451±2.026）pg／mL,（23.918±0.369）pg／mL vs（27.305±2.125）pg／mL],差异具有统计学意义（P〈0.05）。结论全反式维甲酸诱导骨骼畸形大隐.FGF2表茯情况低干骨骼发育正常的大鼠。     

In utero transmission of Mycoplasma pulmonis in experimentally infected Sprague-Dawley rats.

Genital mycoplasmosis is important as an animal model for the interaction between infectious agents and the host during pregnancy as well as in its own right as a confounding variable affecting research projects in which the rat is used as a model to study reproductive function and physiology. We report the in utero transmission of Mycoplasma pulmonis and the development of placentitis, amnionitis, and mild fetal bronchopneumonia in Sprague-Dawley rats. A minimum of 10 days prior to breeding, specific-pathogen-free female Sprague-Dawley rats were infected by intravaginal inoculation with 3 x 10(7) CFU of M. pulmonis X1048 or with an equal volume of sterile broth. Rats and fetuses were subjected to necropsy at days 11, 14, and 18 of gestation. M. pulmonis was able to invade the placenta, cross the placental barrier, and establish an amniotic fluid infection by gestational day 14. It was isolated from the oropharynx and lungs of fetuses at gestational day 18. The placenta was more frequently colonized than amniotic fluid, followed by the fetal oropharynx and lungs, supporting an ascending route of infection. Histopathological evidence also support an active infection, with lesions compatible with placentitis, amnionitis, and mild fetal bronchopneumonia. M. pulmonis can traverse the placenta, resulting in infection of the amniotic fluid and in utero transmission of the microorganism to the developing fetus.     

7076例孕中期唐氏综合征产前筛查结果临床分析

目的 探讨孕中期唐氏综合征（DS）等筛查对检出胎儿出生缺陷和不良妊娠结局的实用价值.方法 用时间分辨荧光分析法对日照市7076名15 ～20孕周孕妇的血清甲胎蛋白（AFP）和游离绒毛膜促性腺激素（F-βHCG）进行检测,结合孕妇年龄、体重、孕周等因素,利用配套孕期胎儿唐氏综合征产前筛查分析软件,分析胎儿患DS、开放性神经管缺陷（NTD）、18-三体综合征的发病风险率,并对高风险孕妇行羊水胎儿细胞染色体核型分析或B超跟踪检查.结果 7076名孕妇中筛出高风险孕妇396例,阳性率为5.6％,在接受羊水检查的56名DS高风险孕妇中,检出唐氏胎儿3例,死胎3例,其它异常3例;对57例神经管缺陷高危孕妇进行B超检查,发现1例无脑儿;在43例18-三体高危的孕妇中,6例做羊水染色体检查,结果均正常.结论 孕中期以唐氏综合征（Ds）产前筛查作为对胎儿先天缺陷,尤其是胎儿染色体异常的筛查是行之有效方法,筛查结果呈高危孕妇须进行羊水染色体核型分析或B超检查.     

Amniotic fluid folate, vitamin B12 and transcobalamins in neural tube defects

Levels of folate, vitamin B12, the vitamin B12 binding proteins, apotranscobalamin I, II and III (TC I, II and III) and the unsaturated vitamin B12 binding capacity (UBBC) were measured in mid-trimester amniotic fluids from normal pregnancies, and from those where the fetus had open spina bifida, anencephaly or omphalocoele, and where the fetus was normal but the mother had had a previous neural tube defect pregnancy. At 15-19 weeks' gestation, vitamin B12 levels were low in the fluids of all the types of abnormal fetuses, and also of normal fetuses where there had been a previous NTD sib. In contradistinction, TC I, II and III and UBBC levels were generally abnormally high in all these groups. Low vitamin B12 levels in the face of high carrier protein levels suggest deranged vitamin B12 production or transport. Since these abnormalities are present in fluids from normal sibs of NTD individuals as well as from those with midline lesions, an inherited defect is implied. We propose that at least part of the genetic predisposition to NTD, and possibly other midline defects, could reside in an abnormality connected with vitamin B12 production, transport or metabolism, and a mechanism is suggested.     

孕妇妊娠中期羊水中Ins13水平研究

目的动物实验得知睾丸间质细胞激素胰岛素样因子3（insulin-like factor 3,Ins13）在腹部睾丸迁移过程中起到非常重要的作用。此过程在人类发生在妊娠中期（第二个3个月）,本研究探讨是否人体中Ins13在睾丸下降过程中也起到一定作用。方法经羊膜穿刺术60名孕妇取羊水测定Ins13及睾酮（testosterone,T）水平,选取样本孕周15-25w,确定胎儿男性28例,女性32例。结果 28例孕男性胎儿羊水中均检测到Ins13（160.5±85.2pg/ml）,而在孕女性胎儿羊水中均未检测到此激素水平。T水平在男性胎儿（1.05±0.30 nmol/L）与女性胎儿（0.33±0.05 nmol/L）相比具有显著差异性（P〈0.0001）。在男性胎儿中Ins13与T阴性相关,Ins13与孕龄统计学阴性相关处于基线水平（P=0.06）,T与孕龄不相关（P=0.10）;相反女性胎儿T水平与孕龄相关（P=0.03）。结论 Ins13存在于人类孕中期孕有男性胎儿羊水中,而孕中期正值睾丸移位过程中。相反在女性胎儿羊水中未检测到Ins13,结果认为Ins13在人类睾丸下降过程同样起到重要作用。     

Date of conception and prevention of neural tube defects

The authors studied the time of conception of 280 fetuses and liveborns with anencephaly or spina bifida seen in a French Canadian population during a period of six years. There is an evident seasonal variation in the conception of NTD fetuses. Using a wave statistical model applied to data recorded over a six year period the authors have rejected the "no variation between the six two-month intervals" hypothesis at a P less than or equal to 0.005 level. It is postulated that, in any vitamin intake or environmental program to prevent NTD in families at risk, one must take into account the annual and seasonal variations, ethnic groups identity and the date of conception in the choice of control mothers.     

大鼠羊水栓塞后类胰蛋白酶、TNF-α水平变化的研究

目的探讨类胰蛋白酶、TNF-α在羊水栓塞中的变化及病理生理作用。方法精选雌性鼠30只于妊娠20天制作羊水栓塞模型。根据注入液体性质不同随机分为对照组（10只）、羊水组（10只）、胎粪液组（10只）。对照组除注入生理盐水外,皆同其余组。实验后取鼠左肺行H.E染色光镜下观察,采用专性底物对模型鼠的血清类胰蛋白酶酶活力进行测定。免疫法检测血中肿瘤坏死因子（TNF-α）含量。结果 1.实验组肺组织可见大量炎性细胞浸润,包括白细胞（主要为中性粒细胞）、巨噬细胞及少量淋巴细胞。2.生理盐水组注入前后类胰蛋白酶水平无明显差异（P〉0.05）;鼠原羊水组和胎粪液组注入后类胰蛋白酶水平明显升高,差异显著（P〈0.01）。3、实验组中TNF-α含量较对照组高,差异显著（P〈0.01）。结论肥大细胞脱颗粒释放类胰蛋白酶可能是羊水栓塞的重要原因,在此基础上引起中性粒细胞、巨噬细胞浸润及一系列病理变化,由此引起肺损伤及临床症状。该酶学方法灵敏、有效,在羊水栓塞临床早期诊断中有一定的应用价值。     

应用SELDI-TOF-MS筛选神经系统先天畸形胎儿羊水诊断标志物

目的分析神经系统先天畸形胎儿羊水和神经系统正常胎儿羊水的蛋白质表达差异。方法选择8例神经系统畸形胎儿羊水,其中男性4例,女性4例;孕妇年龄21～32岁,平均年龄24.9岁;孕周19～34周,平均孕周23.1周。30例经超声检查神经系统正常胎儿羊水作为对照组,其中男性15例,女性15例;孕妇年龄22～29岁,平均年龄25.1岁;孕周19～22周,平均孕周21.2周。运用表面增强激光解吸离子化飞行时间质谱(SELDI-TOF-MS)蛋白质芯片技术检测神经系统先天畸形胎儿羊水和神经系统正常胎儿羊水蛋白质表达图谱。胎儿羊水用WCX2(弱阳离子交换)芯片检测,采用PBSⅡC型蛋白质芯片阅读机读取数据,Protein-Chip software 3.1软件采集数据,Biomarker Wizard软件分析两组羊水的蛋白质差异。结果 SELDI-TOF-MS技术检测发现神经系统先天畸形胎儿羊水和神经系统正常胎儿羊水的蛋白质存在差异表达,共有9个蛋白质水平发生变化,其中质/荷比4 967.526、5 258.056、11 717.010的差异蛋白质在神经系统异常组表达下调,2 540.415、3 107.119、3 396.759、4 590.965、5 589.200、6 429.417的差异蛋白质在神经系统异常组表达上调。结论神经系统先天畸形胎儿羊水和神经系统正常胎儿羊水之间的蛋白质谱有差异蛋白质表达,检测到9个代表性的差异蛋白质很可能是神经系统疾病胎儿的羊水特异性生物标志物。     

Effects of vitamin E on pathological changes induced by diabetes in rat lungs

Twenty-fourmale rats were divided into three groups: Control (C), non-treated diabetic (NTD), and vitamin E-treated diabetic (VETD) groups. After 6 weeks, we evaluated the changes in the alveolar epithelium, alveolar septum thickness, Hcy, and cathepsin G levels in the lung tissue and plasma serine protease inhibitor levels.The results revealed a significant increase in alveolar septum thickness, a high number of type II pneumocytes, high number of glycogen granules, increased vascular elastic membrane thickness, and increased Hcy and cathepsin G levels in the diabetic rats. Plasma level of serine protease inhibitors showed a significant decrease in the NTD animals. The vitamin E-treated rats showed significant amelioration of lung tissue changes, as well as restoration of high cathepsin G, Hcy levels, and serine protease inhibitors when compared to the control rats. These results suggest that diabetes induces lung tissue changes that may be stimulated by Hcy and cathepsin G mediated oxidative stress by, and protective effect could be achieved by using vitamin E.     

Pancreatic amylase expression in human pancreatic development

Enzymatic and immunologic methods were used to study amylase expression in amniotic fluid and human pancreatic tissue from fetuses of various gestational ages. A starch-coated slide assay was used to quantitate amylase activity in amniotic fluids, and samples with activity were studied by electrophoresis to determine the presence of salivary amylase (Amy1) and pancreatic amylase (Amy2) isozymes. Immunohistochemical studies were performed with both a rabbit anti-human Amy1 antibody and a murine anti-human Amy2 antibody (Amy2/SP2/1). Amy2/SP2/1 was used to discriminate between Amy1 and Amy2. Both Amy1 and Amy2 activities were present in amniotic fluid from 14 weeks' gestation, and immunologic activity was present in pancreatic tissue sections from 15.9 weeks' gestation.     

Amniotic fluid can act as an appetitive unconditioned stimulus in preweanling rats

Studies in humans and animals indicate that exposure to flavors in the amniotic fluid during the later gestational period may induce preferences for those flavors. Considering that during the last prenatal period the amniotic fluid contains substances that activate the opioid system, and that this system plays a critical role in the acquisition of olfactory preferences early in life, it has been hypothesized that the amniotic fluid may acquire appetitive unconditioned properties during this period. This has been tested in an experiment in which preweanling rats were exposed to alcohol odor (CS) paired or unpaired with the intraoral infusion of amniotic fluid (US) collected on gestational day 20. The pairing of these two stimuli induced an enhanced palatability of alcohol's flavor as well an increased intake of the drug. These results support the idea that amniotic fluid acquires appetitive unconditioned properties during the last days of gestation and suggest that associative mechanisms involving the amniotic fluid could be underlying odor and taste preferences acquired through fetal exposure.     

Rat strains differ in susceptibility to maternal and fetal infection with Mycoplasma pulmonis

PROBLEM: Vaginally infected Sprague-Dawley (SD) rats are more susceptible to adverse pregnancy outcomes than Wistar (WIS) rats. We postulated that SD rats have enhanced hematogenous spread of Mycoplasma pulmonis to fetal tissues. METHOD OF STUDY: WIS and SD dams were infected intravenously with 10(7), 10(6), and 10(5) colony-forming units of M. pulmonis at gestation day 14. Dams and six randomly selected fetuses were cultured at days 15, 16, 17, and 18 of gestation. RESULTS: In the high-dose group, 100% of fetuses were colonized regardless of rat strain. Significantly higher numbers of M. pulmonis were isolated from placenta (low dose, P < 0.0001; medium dose, P < 0.024; high dose, P < 0.0001), amniotic fluid (low dose, P < 0.003; medium dose, P < 0.017), and fetuses (low dose, P < 0.0011) of SD rats. Spread of M. pulmonis to the amniotic fluid and fetus occurred 1 day earlier in SD rats. CONCLUSIONS: The difference in susceptibility between the two rat strains cannot be explained by hematogenous spread alone. The relative resistance to adverse pregnancy outcomes in WIS rats may be a function of a more robust innate immune system. These rat strains may represent an animal model to address host resistance factors to intrauterine infection.     

Experimental in utero infection of fetal pigs with a porcine parvovirus.

In utero infection of fetuses of six specific-pathogen-free large white sows at 35, 48, 55, 72, 99, and 105 days was studied. The fetuses were infected by direct inoculation of porcine parvovirus into the amniotic sac. The inoculation consisted of 0.25 ml of tissue culture fluid containing 10(5.5) mean tissue culture infective doses per ml of porcine parvovirus strain G10/1. Fetuses of one uterus horn were infected, whereas fetuses in the opposite horn were given 0.25 ml of noninfected cell culture material. No clinical signs of infection were observed; however, all sows developed antibodies 7 to 9 days postinfection. A total of 24 virus-inoculated fetuses and 20 control fetuses were studied. Fetuses infected at 35, 48, and 55 days of gestation died between about 5 and 22 days after infection. Virus was isolated from their organs and fetal blood. Virus spread to control fetuses but did not cause death and mummification or stimulate antibody production. Fetuses from sows infected at 72, 99, and 105 days of gestation survived. They developed high antibody titer in utero. Control piglets remained antibody free.     

蓖麻油引产餐提取物影响子宫收缩和羊膜组织前列腺素E2的含量

背景：有研究显示蓖麻籽提取物具有一定的抗生育作用,然而国内对蓖麻籽在抗生育方面的报道很少。 目的：探讨蓖麻油引产餐提取物对大鼠子宫收缩和羊膜组织前列腺素E2含量的影响。 方法：选取60只Wistar未孕成年雌性大鼠,随机分为生理盐水组、缩宫素组及引产餐提取物组,各20只,观察并记录给药前后各组大鼠离体子宫的收缩强度、频率及活动力。选取90只妊娠18 d的Wistar成年雌性大鼠,随机分为生理盐水组、缩宫素组及引产餐提取物组,各30只,利用放射免疫方法测定给药后各组大鼠羊膜组织前列腺素E2水平。取生理盐水组大鼠羊膜组织,体外培养羊膜细胞,在培养液中加入不同质量浓度蓖麻酸,培养18 h,利用放射免疫方法测定羊膜细胞前列腺素E2水平。 结果与结论：与给药前及生理盐水组相比,给药后引产餐提取物组大鼠子宫的收缩强度、频率及活动力均明显提高,与缩宫素组相比,给药后引产餐提取物组大鼠子宫的收缩强度、频率差异无显著性意义(t=2.321,    P > 0.05),而活动力较低(t=2.765,P < 0.05);与生理盐水组相比,引产餐提取物组妊娠大鼠羊膜组织前列腺素E2水平显著增高(t=14.91,P < 0.01),但引产餐提取物组妊娠大鼠羊膜组织前列腺素E2水平低于缩宫素组(t=2.769,P < 0.05)。大鼠羊膜细胞前列腺素E2水平与蓖麻酸质量浓度及培养时间呈正相关。提示蓖麻油引产餐提取物能够增强宫缩效果、增加羊膜组织前列腺素E2水平,蓖麻酸可能是蓖麻油引产餐的活性成分。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程      

Granulocyte-macrophage colony-stimulating factor levels in amniotic fluid before the onset of labor and during labor do not differ in normal pregnancies

PROBLEM: Granulocyte-macrophage colony-stimulating factor (GM-CSF) at the implantation site may regulate invasion and differentiation of placental trophoblast. We evaluated whether GM-CSF levels in amniotic fluid during labor contributing to subsequent delivery differed from those before the onset of labor in normal pregnancies. METHOD OF STUDY: This study enrolled 36 Japanese women experiencing normal pregnancies with single fetuses who had no infection. Of these pregnancies, 18 were women during labor that led to subsequent term delivery (labors). The other 18 were women without labor underwent cesarean section (controls). These two groups (18 labors and 18 controls) were compared. The average gestational age at entry was 38-39 weeks of gestation. The women's ages and gestational ages did not differ significantly between the two groups. Amniotic fluid was collected and the GM-CSF levels were compared between two groups. The GM-CSF level was determined by the enzyme-linked immunosorbent assay method. RESULTS: There was no significant increase in GM-CSF levels in amniotic fluid during labor compared with that before the onset of labor. CONCLUSIONS: The GM-CSF in amniotic fluid may not promote the onset of labor at term and/or term labor contributing to subsequent delivery may not induce the production and secretion of GM-CSF into amniotic cavity.     

Macrophage colony-stimulating factor levels in amniotic fluid before and after the onset of labor do not differ in normal pregnancies

PROBLEM: Macrophage colony-stimulating factor (M-CSF) promotes placental growth and maintenance. M-CSF also regulates trophoblast invasion into the placental bed. We evaluated whether M-CSF levels in amniotic fluid during labor contributing to subsequent delivery differed from those before the onset of labor in normal pregnancies. METHOD OF STUDY: This study enrolled 48 Japanese women experiencing normal pregnancies with single fetuses who had no infection. Of these pregnancies, 24 were women during labor: 22 led to subsequent term delivery (labors); two had premature delivery. The other 24 were women without labor underwent cesarean section (controls). These two groups (22 labors and 24 controls) were compared. The average gestational age at entry was 38 weeks of gestation. The women's ages and gestational ages did not differ significantly between the two groups. Amniotic fluid was collected and the M-CSF levels were compared between two groups. The M-CSF level was determined by the sandwich enzyme-linked immunosorbent assay (ELISA) method. RESULTS: The levels of M-CSF in amniotic fluid did not differ significantly between the women during labor and those without labor. CONCLUSIONS: M-CSF in amniotic fluid may not contribute to the onset of labor in term pregnancy and/or labor resulting in subsequent delivery may not induce the production and secretion of M-CSF into amniotic cavity.     

Association of combined GIF290T>C heterozygous mutation/FUT2 secretor variant with neural tube defects

Folate and vitamin B12 are needed for the proper embryo‐fetal development possibly through their interacting role in the 1‐carbon metabolism. Folate fortification reduces the prevalence of complex birth defects, and more specifically neural tube defects (NTDs). GIF and FUT2 are 2 genes associated with the uptake and blood level of vitamin B12. We evaluated GIF and FUT2 as predictors of severe birth defects, in 183 aborted fetuses compared with 375 healthy newborns. The GIF290C allele frequency was estimated to 0.4% in healthy newborns and to 8.1% in NTD fetuses (odds ratio 17.8 [95% confidence interval CI: 4.0‐77.6]). The frequency of FUT2 rs601338 secretor variant was not different among groups. The GIF 290C heterozygous/FUT2 rs601338 secretor variant combined genotype was reported in 6 of the 37 NTD fetuses, but not in other fetuses and healthy newborns (P < .0001). This GIF/FUT2 combined genotype has been previously reported in children with congenital gastric intrinsic factor (GIF) deficiency, with respective consequences on B12 binding activity and GIF secretion. In conclusion, a genotype reported in congenital GIF deficiency produces also severe forms of NTD. This suggests that vitamin B12 delivery to neural tissue by the CUBN/GIF pathway could play a role in the neural tube closure mechanisms.     

Increased levels of apo-transcobalamins I and II in amniotic fluid from pregnant women with previous neural tube defect offspring

In an attempt to identify biochemical components of the genetic predisposition to neural tube defects (NTDs), levels of folate, cobalamin, apo-transcobalamins I and II and alpha-fetoprotein were studied in midtrimester amniotic fluid from 24 pregnant women who had previously had a child with NTD. The control group consisted of 76 mothers, subjected to amniocentesis for reasons other than risk of NTD in offspring. Only pregnancies with normal outcome were included. No differences were found between groups for levels of folate, cobalamin or alpha-fetoprotein. Folate intake or metabolism did not appear to differ between groups. In contrast, the level of apo-transcobalamin I was doubled and the level of apo-transcobalamin II tripled in amniotic fluid from women who had had a child with NTD compared with the control group. Since the variation in apo-transcobalamin II in adults is to a high degree genetically determined, the present results may suggest that the genetic predisposition to NTD is associated with variation in this protein. Further studies are needed to substantiate or reject this possibility.