Serum Phoenixin-14 levels of women with polycystic ovary syndrome increase proportionally with BMI.

OBJECTIVE: This study aimed to compare serum PNX-14 values of women with PCOS classified as lean or overweight according to the BMI values. PATIENTS AND METHODS: Fifty lean or overweight women diagnosed with PCOS according to the revised Rotterdam criteria were included in the study. They were divided into two groups according to their BMI values. Thirty patients with BMI values of 18.5-24.9 kg/m2 constituted the normal-weight PCOS group. Twenty patients with BMI values of 25-29.9 kg/m2 formed the overweight PCOS group. Thirty patients with regular menstrual cycles who did not exhibit clinical and laboratory findings of PCOS were selected as the control group. The patients in the control group were also divided into two different groups as normal weight (n=17) and overweight (n=13). In anovulatory PCOS group, blood samples were collected on the third day of progesterone withdrawal bleeding. Both in ovulatory PCOS and control groups, blood samples were collected on the third day of spontaneous menstrual cycle. In addition to basal hormonal parameters, serum phoenixin-14 concentrations were measured by enzyme-linked immunosorbent assay. RESULTS: LH values of the overweight or lean PCOS group were significantly higher than the overweight or lean non-PCOS group (p<0.01). The LH/FSH ratios of the lean and obese PCOS groups were significantly higher than the non-PCOS control group (p<0.01). Testosterone levels of both lean and obese PCOS groups were significantly higher than non-PCOS groups (p<0.02). The HOMA-IR value of the obese PCOS group was significantly higher than the lean PCOS group (p<0.03). The HOMA-IR values of the patients in the PCOS group were significantly higher than the non-PCOS controls. Phoenixin-14 levels of the obese PCOS group were approximately three times higher than the lean PCOS group (p<0.01). Phoenixin-14 levels of the obese non-PCOS group were also three times higher than the lean non-PCOS group (p<0.01). Serum Phoenixin-14 levels of patients in the lean PCOS group were significantly higher than in the lean non-PCOS group (9.11±2.09 pg/mL vs. 2.04±0.11 pg/mL, p<0.01). Serum Phoenixin-14 levels of the patients in the obese PCOS group were significantly higher than in the obese non-PCOS group (27.4±3.04 pg/mL vs. 6.44±1.09 pg/mL, p<0.01). A positive and significant correlation was found between serum PNX-14 levels and BMI, HOMA-IR, LH, and testosterone levels in both lean and obese PCOS patients. CONCLUSIONS: This study showed for the first time that serum PNX-14 levels are significantly increased in lean and obese PCOS patients. The increase in PNX-14 showed a proportional trend with BMI levels. Serum PNX-14 levels were correlated positively with serum LH, testosterone, and HOMA-IR.

Hydrosalpingeal fluid leads to subfertility via endometrial TNF-α and IL-7 and NF-κB signaling pathways.

OBJECTIVE: To determine the concentrations of the nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α) and interleukin-7 (IL-7) in midluteal phase endometrial samples of infertile patients diagnosed with uni or bilateral hydrosalpinx (HX). PATIENTS AND METHODS: A total of 24 patients who decided to undergo laparoscopic salpingectomy were included in the study. Salpingectomy indications consisted of patients with a diagnosis of hydrosalpinx (n=12) or ectopic pregnancy (n=12). Twelve healthy patients who underwent Pomeroy-type tubal ligation were considered as the second and healthy control group. The diagnosis of hydrosalpinges was made by transvaginal 2D ultrasonography or HSG. All patients in the hydrosalpinges or ectopic pregnancy group underwent laparoscopic salpingectomy. Just before salpingectomy, endometrial samples were obtained from all patients by Pipelle cannula. Endometrial sampling was performed 7-9 days after the LH surge in the control group. IL-7, NF-κB and TNF-α concentrations were measured by ELISA method in the endometrial samples of all three groups. RESULTS: The endometrial IL-7 concentration before salpingectomy of the patients in the hydrosalpinx group was 44.6±6.65 ng/mg wet-tissue. The IL-7 levels of the HX group were significantly higher than those of the patients in the ectopic pregnancy group (19.3±3.06 ng/mg wet tissue versus 44.6±6.65 ng/mg wet tissue, p<0.04). Similarly, IL-7 levels of the HX group were significantly higher than those of the tubal ligation group (6.08±1.48 ng/mg wet tissue versus 44.6±6.65 ng/mg wet tissue, p<0.03). The endometrial TNF-α concentration of the patients in the hydrosalpinx group was 33.20±5.40 ng/mg wet-tissue. The TNF-α value detected in the hydrosalpinx group was significantly higher than both the TNF-α value in the ectopic pregnancy group (11.8±1.07 ng/mg wet-tissue vs. 33.20±5.40 ng/mg wet-tissue, p<0.01) and the TNF-α value in the tubal ligation group (5.30±1.22 ng/mg wet-tissue vs. 33.20±5.40 ng/mg wet-tissue, p<0.01). The pre-salpingectomy endometrial NF-κB concentration of the patients in the hydrosalpinx group was 6.38±1.40 ng/mg wet-tissue. This value is higher than endometrial NF-κB levels in the ectopic pregnancy group (3.67±0.41 ng/mg wet-tissue vs. 6.38±1.40 ng/mg wet-tissue, p<0.02) and NF-κB levels in the tubal ligation group (1.07±0.38 ng/mg wet-tissue vs. 6.38±1.40 ng/mg wet-tissue, p<0.01). CONCLUSIONS: The presence of hydrosalpinx prevents successful implantation by increasing the levels of endometrial proinflammatory cytokines TNF-α, IL-7 and NF-κB.

The efficacy of a simple single-paged visual illustration on anxiety during pediatric gastroscopy: a randomized clinical trial.

BACKGROUND AND STUDY AIMS: Pediatric gastroscopy is a widely used invasive procedure and causes significant anxiety. In this prospective trial, the efficacy of a new original single-page illustration in reducing anxiety was evaluated. PATIENTS AND METHODS: One hundred fifty-two consecutive children aged between 7 and 18 years who were scheduled to undergo gastroscopy were randomized to receive a standard consent form plus the new single-page visual illustration (group I) and the usual text consent brochure (group II). The Hospital Anxiety and Depression Scale (HADS) was administered to mothers just before, at the first hour, and at the second week after gastroscopy. Also, Visual Analogue Scale (VAS) scores were obtained from children to measure anxiety at the same time intervals. RESULTS: The demographic characteristics of the groups were similar. A higher number of cases refused gastroscopy in group II (p=0.040). The proportion of mothers with significant HADS- Anxiety scores at the baseline evaluation (≥8) was 33.8% in group I, whereas it was 58.1% in group II (p=0.003). This remarkable difference in anxiety was also observed at the first hour and second week. Moreover, significant baseline HADS-Depression scores (≥8) of the mothers were more common in group II (2.7% vs. 12.7%, p=0.028). In addition, overall HADS scores revealed considerably higher values in group II at all time intervals. VAS scores in children revealed significantly less anxiety in group I at both the baseline and the first hour assessment. CONCLUSIONS: Lower anxiety scores were observed in both mothers and children with the incorporation of the single-page illustration. This new modality may provide less anxiety and prevent insufficient comprehension with higher acceptance of gastroscopy.

ROSACEA IS ASSOCIATED WITH THYROID AUTOIMMUNITY: A CASE CONTROL STUDY.

BACKGROUND AND AIMS: There are numerous intrinsic and extrinsic factors associated with the pathophysiology of rosacea, including immune dysregulation. The aim of this study is to determine the relationship between rosacea and thyroid autoimmunity. METHODS: Seventy-two patients with rosacea and 62 rosacea-free control subjects were included in the study. Serum free triiodothyronine (fT3), free thyroxine (fT4), thyrotropin releasing hormone (TSH), antithyroidperoxidase antibody (ATPO), antithyroglobulin (anti-Tg), prolactin, dehydroepiandrosterone sulfate (DHEAS), basal cortisol, serum CRP concentrations and erythrocyte sedimentation rate were measured. RESULTS: The number of the cases with high levels of CRP, anti-M and prolactin in rosacea group were significantly higher than the controls (p<0.05), there was no significant difference according to other parameters in both groups (p>0.05). There was no significant difference according to the presence of a thyroid disease in both groups (p>0.05). The decrease in the CRP parameter in patients with the disease duration of 1-5 years was found to be statistically significant (ßCRP=-0.251, pCRP<0.05). There was no statistically significant difference according to disease severity or disease duration (p>0.05). CONCLUSIONS: Rosacea may be associated with high thyroid autoantibodies, prolactin and CRP levels, in which immune-endocrine interactions are important.

SEEG dipole source localization based on an empirical Bayesian approach taking into account forward model uncertainties.

Electromagnetic brain source localization consists in the inversion of a forward model based on a limited number of potential measurements. A wide range of methods has been developed to regularize this severely ill-posed problem and to reduce the solution space, imposing spatial smoothness, anatomical constraint or sparsity of the activated source map. This last criteria, based on physiological assumptions stating that in some particular events (e.g., epileptic spikes, evoked potential) few focal area of the brain are simultaneously actives, has gained more and more interest. Bayesian approaches have the ability to provide sparse solutions under adequate parametrization, and bring a convenient framework for the introduction of priors in the form of probabilistic density functions. However the quality of the forward model is rarely questioned while this parameter has undoubtedly a great influence on the solution. Its construction suffers from numerous approximation and uncertainties, even when using realistic numerical models. In addition, it often encodes a coarse sampling of the continuous solution space due to the computational burden its inversion implies. In this work we propose an empirical Bayesian approach to take into account the uncertainties of the forward model by allowing constrained variations around a prior physical model, in the particular context of SEEG measurements. We demonstrate on simulations that the method enhance the accuracy of the source time-course estimation as well as the sparsity of the resulting source map. Results on real signals prove the applicability of the method in real contexts.

Adaptive divergence in embryonic thermal plasticity among Atlantic salmon populations.

In the context of global changes, the long-term viability of populations of endangered ectotherms may depend on their adaptive potential and ability to cope with temperature variations. We measured responses of Atlantic salmon embryos from four populations to temperature variations and used a QST -FST approach to study the adaptive divergence among these populations. Embryos were reared under two experimental conditions: a low temperature regime at 4 °C until eyed-stage and 10 °C until the end of embryonic development and a high temperature regime with a constant temperature of 10 °C throughout embryonic development. Significant variations among populations and population × temperature interactions were observed for embryo survival, incubation time and length. QST was higher than FST in all but one comparison suggesting an important effect of divergent selection. QST was also higher under the high-temperature treatment than at low temperature for length and survival due to a higher variance among populations under the stressful warmer treatment. Interestingly, heritability was lower for survival under high temperature in relation to a lower additive genetic variance under that treatment. Overall, these results reveal an adaptive divergence in thermal plasticity in embryonic life stages of Atlantic salmon suggesting that salmon populations may differentially respond to temperature variations induced by climate change. These results also suggest that changes in temperature may alter not only the adaptive potential of natural populations but also the selection regimes among them.

Moderate genetic drift is driven by extreme recruitment events in the invasive mollusk Crepidula fornicata.

Effective population size (Ne) is a measure of genetic drift and is thus a central parameter in evolution, conservation genetics and invasion biology. Interestingly, in native marine species, Ne is typically several orders of magnitude lower than the census size. This pattern has often been explained by high fecundity, variation in reproductive success and pronounced early mortality, resulting in genetic drift across generations. Data documenting genetic drift and/or Ne in marine invasive species are, however, still scarce. We examined the importance of genetic drift in the invasive species Crepidula fornicata by genotyping 681 juveniles sampled during each annual recruitment peak over nine consecutive years in the Bay of Morlaix (Brittany, France). Observed variations in genetic diversity were partially explained by variation in recruitment intensity. In addition, substantial temporal genetic differentiation was documented (that is, genetic drift), and was attributed to nonrandom variance in the reproductive success of different breeding groups across years in the study species. Using a set of single-sample and temporal estimators for Ne, we estimated Ne to be three or four orders of magnitude smaller than the census size (Nc). On one hand, this reduction in Ne relative to Nc appeared congruent with, although slight higher than, values commonly observed in native marine species. Particular life-history traits of this invasive species may play an important role in buffering genetic drift. On the other hand, Ne still remained far below Nc, hence, possibly reducing the efficiency of selection effects.

The use of human hair as biodosimeter.

The potential use of human hair samples as biologic dosimeter was investigated by electron spin resonance (ESR) spectroscopy. The hair samples were obtained from female volunteers and classified according to the color, age and whether they are natural or dyed. Natural black, brown, red, blonde and dyed black hair samples were irradiated at low doses (5-50Gy) and high doses (75-750Gy) by gamma source giving the dose rate of 0.25Gy/s in The Sarayköy Establishment of Turkish Atomic Energy Authority. While the peak heights and g-values (2.0021-2.0023) determined from recorded spectra of hair were color dependent, the peak-to-peak line widths were varied according to natural or dyed hair (ΔHpp: 0.522-0.744mT). In all samples, the linear dose-response curves at low doses saturated after ~300Gy. In black hair samples taken from different individuals, differences in the structure of the spectrum and signal intensities were not observed. The EPR signal intensities of samples stored at room temperature for 22 days fell to their half-values in 44h in black hair, 41h in blonde and brown hairs, 35h in dyed black hair and in 17h in red hair. The activation energies of samples annealed at high temperatures for different periods of time were correlated well with those obtained in the literature. In conclusion, hair samples can be used as a biological dosimeter considering the limitations showed in this study.

Evaluating dipolar source localization feasibility from intracerebral SEEG recordings.

Stereo-electroencephalography (SEEG) is considered as the golden standard for exploring targeted structures during pre-surgical evaluation in drug-resistant partial epilepsy. The depth electrodes, inserted in the brain, consist of several collinear measuring contacts (sensors). Clinical routine analysis of SEEG signals is performed on bipolar montage, providing a focal view of the explored structures, thus eliminating activities of distant sources that propagate through the brain volume. We propose in this paper to exploit the common reference SEEG signals. In this case, the volume propagation information is preserved and electrical source localization (ESL) approaches can be proposed. Current ESL approaches used to localize and estimate the activity of the neural generators are mainly based on surface EEG/MEG signals, but very few studies exist on real SEEG recordings, and the case of equivalent current dipole source localization has not been explored yet in this context. In this study, we investigate the influence of volume conduction model, spatial configuration of SEEG sensors and level of noise on the ESL accuracy, using a realistic simulation setup. Localizations on real SEEG signals recorded during intracerebral electrical stimulations (ICS, known sources) as well as on epileptic interictal spikes are carried out. Our results show that, under certain conditions, a straightforward approach based on an equivalent current dipole model for the source and on simple analytical volume conduction models yields sufficiently precise solutions (below 10mm) of the localization problem. Thus, electrical source imaging using SEEG signals is a promising tool for distant brain source investigation and might be used as a complement to routine visual interpretations.

Population differences in response to hypoxic stress in Atlantic salmon.

Understanding whether populations can adapt to new environmental conditions is a major issue in conservation and evolutionary biology. Aquatic organisms are increasingly exposed to environmental changes linked with human activities in river catchments. For instance, the clogging of bottom substratum by fine sediments is observed in many rivers and usually leads to a decrease in dissolved oxygen concentrations in gravel beds. Such hypoxic stress can alter the development and even be lethal for Atlantic salmon (Salmo salar) embryos that spend their early life into gravel beds. In this study, we used a common garden experiment to compare the responses to hypoxic stress of four genetically differentiated and environmentally contrasted populations. We used factorial crossing designs to measure additive genetic variation of early life-history traits in each population. Embryos were reared under normoxic and hypoxic conditions, and we measured their survival, incubation time and length at the end of embryonic development. Under hypoxic conditions, embryos had a lower survival and hatched later than in normoxic conditions. We found different hypoxia reaction norms among populations, but almost no population effect in both treatments. We also detected significant sire × treatment interactions in most populations and a tendency for heritability values to be lower under stressful conditions. Overall, these results reveal a high degree of phenotypic plasticity in salmon populations that nevertheless differ in their adaptive potential to hypoxia given the distinct reaction norms observed between and within populations.

Determination of major and minor elements in the Malva sylvestris L. from Turkey using ICP-OES techniques.

In this work, Malva sylvestris var. mauritiana (L.) leaves were collected from different points in Muradiye region of Manisa-Turkey. The leaves were dissolved by wet digestion method using a mixture of mineral acid. Concentrations of Ag, Al, B, Ba, Bi, Ca, Cd, Co, Cr, Cu, Fe, K, La, Mg, Mn, Na, Ni, Pb, Sn, Sr, Sb, Si, Ti, U, Zn, and Zr in prepared solutions were determined by using inductively coupled plasma optical emission spectrometry (ICP-OES). High Ca (13,848 mg/kg) and Mg (1,936 mg/kg) concentrations were found at the leaves. Obtained values were compared with the internationally permitted (standard) values. The results of elements were analyzed statistically (analysis of variance test). For different leaf sizes, concentration factors were calculated.

Peripheral blood lymphocytes from patients with rheumatoid arthritis are differentially sensitive to apoptosis induced by anti-tumour necrosis factor-alpha therapy.

OBJECTIVE: The efficacy of anti-tumour necrosis factor-alpha (TNF-alpha) therapies in rheumatoid arthritis (RA) has been mainly attributed to TNF-alpha neutralisation. Other mechanism as immune cell apoptosis, which is impaired in RA, may also be induced by anti-TNF-alpha therapies. The aim of our study was to investigate whether TNF-alpha inhibitors could induce apoptosis in vitro of the peripheral blood lymphocytes of RA patients. METHODS: Peripheral blood mononuclear cells (PBMC) isolated from 24 patients with RA and 18 healthy donors were incubated with anti-TNF-alpha agents, infliximab or etanercept, in comparison with no agent and including an isotypic control, for 48 hours. Apoptosis was detected and quantified by annexin V labelling of phosphatidylserine externalization using cytofluorometric analysis and compared with PBMC production TNF-alpha in vitro. RESULTS: In healthy donors, induced apoptosis was observed in 0.3% to 3.8% of lymphocytes with both therapies. In RA patients the treatment induced lymphocyte apoptosis in 17 of 24 patients with a percentage of annexin V-positive lymphocytes ranging from 0.1% to 25%. Among these 17 RA patients, a significant in vitro lymphocyte apoptosis (> 4%) was observed in 11 patients (46%) compared with healthy donors (p < 0.01). The variability of the response to anti-TNF-alpha within the RA population was not dependent on TNF-alpha synthesis or disease activity. CONCLUSION: In vitro induction of lymphocyte apoptosis by anti-TNF-alpha was observed in a subgroup of RA patients. Based on these data, it would be of interest to further study the interindividual variations of sensitivity to apoptosis induced by TNF alpha inhibitors in relation to treatment efficacy or resistance observed in RA patients.

An assessment of the quality of various bottled mineral water marketed in Turkey.

Fifteen bottled mineral waters purchased at random all over Turkey were analyzed for their chemical composition by OPTIMA-2000 ICP-AES Perkin Elmer techniques. Results show a wide spread in the chemical specification of these mineral waters, with differences in chemical composition observed in the regions being due to the geological environment and the majority of bottled mineral waters exceeding the pH limit of Turkish drinking water standards. When the concentrations of elements are evaluated, it can readily be seen that generally there are three types of mineral water in Turkey. The concentrations of Al, B, Ba, Cd, Cu, Cr, Fe, Mn Pb and Zn in mineral water were compared with the limits established by the Turkish Standard for Natural Mineral Waters (Turkish Official Gazette 2004); water standards prepared by World Health Organization (2006) and the United States of America Environmental Protection Agency (US EPA) drinking water standards (1993). Such a comparison shows that, except for Ba and Mn, the concentrations of the other heavy metals are lower than the limit of the US. EPA in Turkey. Some parameters examined were found to comprise strong correlations pair-wise.

ACE I/D gene polymorphism and aerobic endurance development in response to training in a non-elite female cohort.

AIM: The aim of this study was to investigate the association between ACE gene polymorphism and short- and medium-duration aerobic endurance performance improvements in response to the same training regimen in a non-elite female cohort. METHODS: Fifty-five female non-elite Caucasian Turkish athletes trained to enhance running speeds corresponding to 70% and 90% of heart rate reserve (V-HRR70 and V-HRR90 respectively) 30 min running speed performance (V-30min) 3 times per week, for 6 weeks. ACE gene polymorphisms studied by PCR analysis. RESULTS: The distribution of genotypes in the whole cohort was 21.8%, 41.8%, 36.4% for II (n=12), ID (n=23) and DD (n=20), respectively. Subjects with ACE II genotype had significantly higher improvements in V-30min and V-HRR70 than the ACE DD group (P<0.05). However, in HRR90 ACE DD genotype had a better performance enhancement in running speed than others (P<0.05). Endurance improvements in the V-HRR70 and in the V-30min showed a linear trend as II>ID>DD (P<0.05 and P<0.01, respectively) while a linear trend as DD>ID>II (P<0.01) observed in V-HRR90. CONCLUSION: ACE II genotype may related with better improvements in medium duration aerobic endurance performance whilst ACE DD genotype seems to be more advantageous in performance enhancement in shorter duration and higher intensity endurance activities.

The influence of homocysteine levels on endothelial function and their relation with microvascular complications in T2DM patients without macrovascular disease.

The aim of this study was to investigate the influence of homocysteine (hcy) levels on endothelial function by the method of brachial artery ultrasonography and their relation with microvascular complications in type 2 diabetes mellitus (T2DM) patients without macrovascular disease. Fifty-nine T2DM patients with a mean age of 53.4+/-8.6 years and diabetes duration of 8.1+/-6.2 years and 16 healthy controls with a mean age of 47+/-14.5 years were included in the study. Endothelialdependent and endothelium-independent flow-mediated dilatation (FMD) were evaluated via brachial artery ultrasonography. Fasting plasma glucose (FPG), glycosylated haemoglobin (A1c), lipid profile, hcy, B12 and folic acid levels were measured. Diabetic patients and control group individuals were compared with regard to the laboratory values and brachial artery vascular reactivity. Factors influencing endothelium-dependent FMD were investigated with linear regression analysis. Age, gender, body mass index, lipid profiles and hcy levels were similar in both groups (p>0.05). Endothelium-dependent FMD percentages were significantly lower in diabetics than in the control group (7.7+/-5.9 vs. 11.7+/-7.1%, p<0.05). Endothelial-independent FMD percentage was similar for both groups (p>0.05). The upper limit of the reference hcy value was found to be 12.6 micromol/l in the control group. In the diabetic group, hcy levels were high in 33 patients and normal in 26 patients. No difference was detected between the patients with high hcy levels and those with a normal level with regard to endothelium-dependent and endothelium-independent FMD values (p>0.05). Mean hcy levels were 16+/-1.7 and 13.3+/-4.3 micromol/l in T2DM patients with microvascular complication and those with no microvascular complication, respectively (p<0.05). Regression analysis revealed that the main factors influencing the endothelial-dependent FMD were FPG, total cholesterol (TC), triglycerides (TG) and high-density lipoprotein (HDL-C) levels (p<0.05, p=0.05, p=0.05, p=0.02, respectively). Hcy, folic acid and B12 values did not influence endothelium-dependent FMD (p>0.05). Diabetes duration and A1c levels were close to being significant although they did not reach statistical significance (p=0.07 and p=0.08 respectively). Hcy levels have no effect on endothelium-dependent and endothelium-independent FMD in T2DM patients without macrovascular complications. The influence of classical atherogenic factors (such as FPG, TC, TG and HDL-C levels) on endothelium functions, detected with endothelium-dependent FMD, is greater.

Delayed maturation of immunoglobulin G avidity: implication for the diagnosis of toxoplasmosis in pregnant women.

The low avidity of immunoglobulin G has been reported to be a useful marker of recent infection with Toxoplasma. Several investigators, however, have published discrepant result on the maturation of avidity over time. The aim of this study was to analyse persistent low avidity of immunoglobulin G in immunocompetent individuals and in pregnant women and how it could interfere in the flowchart of antenatal diagnosis of toxoplasmosis in the latter group. An international literature search was conducted together with a retrospective study of a hospital database. Eleven publications that met the inclusion criteria reported delayed maturation of avidity at a frequency ranging from 0 to 66.6% of the patients. Examination of those publications demonstrated an important heterogeneity in the type of assay used, the calculation of avidity, the cutoff above which avidity was considered to be elevated, and the delay since infection after which indices are expected to be high. In the hospital database, persistent low avidity was found even after a median follow-up period of 6 years. Different factors could interfere with maturation of avidity, such as variations between individuals, the assay system used, and, possibly, the treatment administered. The results of this study clearly demonstrate that, in a pregnant woman, an acute infection cannot be reliably diagnosed solely on the basis of low avidity of immunoglobulin G. Further investigations and standardization of assays are urgently needed. Estimation of the time of infection remains difficult, especially in cases in which the samples are drawn late in pregnancy; the final estimate must be based on several tests repeated at intervals of weeks.