Female Serum Samples Research Articles

Secretion of anti-Müllerian hormone in the Florida manatee Trichechus manatus latirostris, with implications for assessing conservation status

Environmental and anthropogenic stressors can affect wildlife populations in a number of ways. For marine mammals (e.g. the Florida manatee Trichechus manatus latirostris), certain stres- sors or conservation risk factors have been identified, but sublethal effects have been very difficult to assess using traditional methods. The development of 'biomarkers' allows us to correlate effects, such as impaired reproduction, with possible causes. A recently developed biomarker (anti-Mullerian hor- mone, AMH) provides an enzyme-linked immunosorbent assay of gonadal function. The study objec- tive was to determine AMH levels in wild manatees. In total, 28 male and 17 female manatee serum samples were assayed. Animal demographics included collection date, body weight (kg) and total length (cm). In certain cases, age of individuals was also known. AMH levels ranged from 160 to 2451.85 ng ml -1 (mean = 844.65 ng ml -1 ) in males and 0.00 to 0.38 ng ml -1 (mean = 0.10 ng ml -1 ) in females. Linear regression analyses revealed a significant relationship between male AMH levels and body weight (R 2 = 0.452; p < 0.001) and length (R 2 = 0.338; p < 0.001). Due to the small sample size, regression analyses for female AMH and body weight and length were not significant. This rep- resents the first report of AMH detection in a marine mammal. AMH levels in male manatees are the highest of any species observed to date, whereas levels in females are within reported ranges. Fur- ther studies will promote improved conservation decision by assessing AMH levels in the manatee as a function of various stressors including, but not limited to, nutritional status, serious injuries (e.g. watercraft collisions), exposure to biotoxins or contaminants, or disease.

Levels of perfluorooctane sulfonate and perfluorooctanoic acid in female serum samples from Japan in 2008, Korea in 1994–2008 and Vietnam in 2007–2008

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have recently received attention owing to their widespread contamination in the environment. One of major manufacturers, 3M Company voluntarily phased out PFOS production in 2002. We measured the PFOS and PFOA concentrations in serum samples from Japan (Sendai, Takayama and Osaka), Korea (Busan and Seoul) and Vietnam (Hanoi) to evaluate the possible effects of the phase-out on the serum levels. There were spatial differences in both the serum PFOS and PFOA concentrations. The serum PFOS concentrations (ng mL −1) evaluated as the geometric mean (geometric standard deviation) in 2007–2008 ranged from 4.86 (1.45) in Sendai, Japan, to 9.36 (1.42) in Busan, Korea. The serum PFOA concentrations ranged from 0.575 (2.32) in Hanoi, Vietnam, to 14.2 (1.73) in Osaka, Japan. Historically archived samples collected from Korea in 1994–2008 revealed that the serum PFOA concentrations increased by 1.24-fold in Busan from 2000 to 2008 and 1.41-fold in Seoul from 1994 to 2007. On the other hand, the serum PFOS concentrations did not change from 1994 to 2007/2008. The serum PFOS levels in Japan in 2008 were significantly decreased compared with previously reported values (22.3–66.7% of the values in 2003/2004). However, the serum PFOA levels showed a clear decline from 2003 to 2008 in a high-exposed area, Osaka, but not in low-exposed areas in Japan. The trends toward decreases were not uniformly observed in Asian countries, unlike the case for the United States, suggesting that local factors associated with the production and introduction histories in each country overwhelm the effects of the phase-out.

Serum Total Prolactin and Monomeric Prolactin Reference Intervals Determined by Precipitation with Polyethylene Glycol: Evaluation and Validation on Common ImmunoAssay Platforms

Macroprolactin is an important source of immunoassay interference that commonly leads to misdiagnosis and mismanagement of hyperprolactinemic patients. We used the predominant immunoassay platforms for prolactin to assay serum samples treated with polyethylene glycol (PEG) and establish and validate reference intervals for total and monomeric prolactin. We used the Architect (Abbott), ADVIA Centaur and Immulite (Siemens Diagnostics), Access (Beckman Coulter), Elecsys (Roche Diagnostics), and AIA (Tosoh) analyzers with samples from healthy males (n = 53) and females (n = 93) to derive parametric reference intervals for total and post-PEG monomeric prolactin. Concentrations of immunoreactive prolactin isoforms in serum samples from healthy individuals were established by gel filtration chromatography (GFC). We then used samples from 22 individuals whose hyperprolactinemia was entirely attributable to macroprolactin and 32 patients with true hyperprolactinemia to compare patient classifications and prolactin concentrations measured by GFC with the newly derived post-PEG reference intervals. Parametric reference intervals for post-PEG prolactin in male and female serum samples, respectively, were (in mIU/L): 61-196, 66-278 (Centaur); 63-245, 75-381 (Elecsys); 70-301, 92-469 (Access); 72-229, 79-347 (Architect); 73-247, 83-383 (AIA); and 78-263, 85-394 (Immulite). Concordance between GFC and immunoassay-specific post-PEG reference intervals was observed in 311 of 324 cases and for 31 of 32 patients with true hyperprolactinemia and 17 of 22 patients with macroprolactinemia. Results leading to misclassification occurred in a few analyzers for 5 macroprolactinemia patient samples with relatively minor increases in post-PEG prolactin (mean 61 mIU/L). Our validated normative reference data for sera pretreated with PEG and analyzed on the most commonly used immunoassay platforms should facilitate the more widespread introduction of macroprolactin screening by clinical laboratories.

Prevalence of Hepatitis B virus genotype D in females in Karachi, Pakistan

Hepatitis B virus (HBV) is an etiological agent of acute and chronic liver disease existing throughout the world. The high genetic variability of HBV genome is reflected by eight genotypes (A to H), and each genotype has characteristic geographical distribution, which is important epidemiologically. Previous studies from the province of Sindh, Pakistan, have reported that genotypes A and D as prevalent HBV genotypes. The aim of the study was to investigate the prevalence of HBV genotypes in physically healthy females at two universities in Karachi, Sindh, Pakistan. Blood was collected from a total of 4,000 healthy female volunteer students and serum samples obtained were screened for Hepatitis B surface antigen (HBsAg), and anti-HBs antibodies by immunochromatography and ELISA. Genotyping was conducted for 6 HBV genotypes (A through F). Both genotyping and sequencing data of HBV positive females are described. Out of 4,000 volunteers, 180 (4.5%) tested positive for HBsAg and 20 (0.5%) were positive for HBs antibodies. All 180 serum samples were genotyped by PCR and sequencing analyses was conducted for 21 samples. Out of 180 HBsAg positive samples, 150 showed a single HBV D genotype infection; 29 showed co-infection of genotypes B and D; and 1 exhibited co-infection of genotypes C and D. Twenty-one representative samples were selected randomly from genotypes B, D, and C for sequencing and each isolate clustered with respective reference genotype sequence, thus validating the genotyping strategy. Genotype D appears to be the dominant genotype prevalent in Karachi's otherwise healthy female population.

O-24: Diphasic pattern of anti-mullerian hormone (AMH) in ovulatory cycles as evidenced by means of an ultra-sensitive assay: New insights into ovarian function

AMH levels have been recognized for a decade as a reliable index of ovarian reserve. However the assay initially developed for testis investigation by Josso et al (JCEM 1990) was designed for the determination of high AMH levels in diluted serum, but was not sensitive enough for the measurement of low AMH levels in diluted samples. On the other hand, a significant matrix effect was observed with neat serum, resulting in underestimation of low AMH levels. As a consequence, the first generation AMH assay was unable to find any significant change in AMH levels during normal menstrual cycles. The aim of the study was to reassess ovarian secretion of AMH by means of a new ultrasensitive assay (DSL, Houston, Texas), suitable for precise and free of matrix effect measurements on diluted female serum samples. AMH as well as other ovarian hormone and gonadotropin levels were measured in women enrolled in a multicenter international trial. They received daily for 3 months either a placebo or the progesterone receptor antagonist VA 2914 (HRA Pharma, Paris, France). The trial was approved by the local Ethics Committee and conducted in compliance with the Declaration of Helsinki The subjects were sampled every 3 days (D) throughout the 3rd cycle of treatment. FSH and LH were measured by means of AutoDelfia reagents (PerkinElmer France), estradiol and progesterone by RIA (DiaSorin-France and CIS biointernational, France, respectively,) and inhibin A by Elisa (Oxford Bioinovation-DSL-France) In 12 placebo treated ovulatory cycles, AMH levels (pmol / l, mean ± sem) exhibited a diphasic pattern, with a peak on D -7 relative to LH peak : 26 ± 3.2, a nadir on D0 : 19.1 ± 3.5 , and a second peak in luteal phase on D10 after LH peak : 25.4 ± 4.3. These variations were significant (p<0.05). There were no correlations between AMH levels and FSH, LH, inhibin A, or progesterone levels. Interestingly AMH levels were negatively correlated with estradiol levels : r = 0.27, p < 0 .02. As expected since both hormones are indexes of luteinization, inhibin A and progesterone levels were strongly correlated : r = 0.62, p <0.001. In 21 anovulatory cycles observed in women treated with the progesterone receptor antagonist, AMH levels exhibited a quite different pattern : no significant changes from D3 to D14 of cycle, significantly higher levels in the second phase of the cycle (D18-D28) : 28.2 ± 2.2 relative to the first phase (D3-D14) : 24.2 ± 1.9, p<0.05. Again no correlations were found between AMH and gonadotropin or ovarian hormone levels. In addition AMH and estradiol levels were not correlated in these anovulatory cycles. It should be noted however that estradiol levels in the second phase of the anovulatory cycles were lower than in luteal phase Thanks to this new AMH assay, we report for the first time a diphasic pattern of AMH levels throughout the menstrual cycle, contrasting with previous reports of unsignificant changes during the cycle. In addition the inverse relationship of estradiol and AMH in ovulatory cycles suggest a possible role of estradiol in the regulation of AMH secretion by the ovary.

Determination of androgen bioactivity in human serum samples using a recombinant cell based in vitro bioassay

The present study describes the development and optimization of a cell-based reporter assay for the determination of androgen bioactivity levels in human serum samples. Towards this end, human embryonic kidney (HEK) 293 cells were cotransfected with two plasmids, one encoding the mouse mammary tumor virus (MMTV)-driven luciferase reporter gene and the other the SV40 promoter-driven human androgen receptor (AR), and a stable cell line, expressing human AR and androgen-responsive luciferase was established by antibiotic selection. RT-PCR confirmed proper transcription and stable integration of AR in the cell line. On stimulation of the cells with testosterone (T) for 24 h, luciferase activity was increased in dose-dependent fashion up to 15-fold, with the minimum effective concentration of T being 0.03 nmol/l. T-induced reporter gene expression of the cells was inhibited by the anti-androgens cyproterone acetate and hydroxyflutamide. Upon stimulation with non-androgenic steroids like estradiol, progesterone, dexamethasone and cortisol, the cells showed only marginal activity except for a weak glucocorticoid effect at high concentration. The cells also responded well to various chemicals (mostly pesticides, their metabolites and common industrial chemicals) with known androgenic activity. The cell line was further optimized by measuring the levels of androgens from male and female serum samples. Our data indicated that the cells can be used to estimate androgen bioactivity in serum of females suffering from polycystic ovary syndrome (PCOS) and males of different age groups. In conclusion, we demonstrate that the bioassay based on this cell line provides a reliable method to determine serum levels of androgen bioactivity from biological samples even at the low concentrations present in female circulation. The 96-well plate format makes the assay suitable for high throughput measurements.

Sex steroid concentrations and localization of steroidogenic enzyme expression in free-ranging female northern fur seals ( Callorhinus ursinus)

Recent precipitous population declines in northern fur seal ( Callorhinus ursinus) and other Alaskan pinniped populations are due in part to reduced fecundity and have emphasized deficits in basic reproductive knowledge of these species. Following estrus and mating, fertilized female pinnipeds experience an obligatory delayed implantation lasting several months and non-pregnant (pseudopregnant) females are indistinguishable by sex steroid levels during this time. The current study examined circulating steroid concentrations and ovarian expression of key steroid-synthesizing enzymes in northern fur seals to identify changes associated with embryonic implantation, data necessary for estimating early pregnancy rates of the population. Blood samples were collected from 84 female fur seals captured on an Alaskan rookery from October 15 to November 30, a period spanning the end of the delay and subsequent uterine implantation of the fertilized blastocyst in this species. Concentrations of progesterone, estradiol, and estrone measured in blood collected during boreal fall were contrasted with samples collected during late summer from post-ovulatory females and males. Serum sex steroids were also related to ovarian expression of key enzymes responsible for androgen (17α-hydroxylase/17,20-lyase, cytochrome b5, and 3β-hydroxysteroid dehydrogenase) and estrogen (aromatase cytochrome P450) synthesis. Enzymes necessary for androgen synthesis were highly expressed in ovaries, and accordingly, endocrine profiles were expanded to include DHEA, androstenediol, androstenedione, and testosterone. Estradiol concentrations were universally low and free and conjugated estrone were the primary circulating estrogens in fur seal sera. No implantation-associated peak was identified for estrogens or progesterone and mean values of progesterone and estrone were actually greater in female serum samples collected during summer than fall. However, there was a significant positive relationship between fall sampling date and testosterone concentration. Additionally, DHEA concentrations in females were lower in samples collected during the implantation period than during the summer embryonic delay. These data suggest androgens may play a substantial regulatory role in the embryonic delay of northern fur seals.

Measurement of total serum testosterone levels using commercially available kits: high degree of between-kit variability

Objective: The measurement of total serum testosterone has an established clinical role in the management of male hypogonadism and female androgen excess disorders. We studied the between-kit variability and precision of six different commercially available testosterone assays and compared them with an established in-house method. Design: Laboratory observational prospective study. Setting: Tertiary university medical center clinical laboratory. Patient(s): Three groups of samples each of men (n = 36) and women (n = 15) who had high, normal, or low levels of sex hormone-binding globulin (SHBG), respectively, were studied. Intervention(s): Individual and pooled (male and female) serum samples were analyzed for total testosterone concentration using six different commercially available assays and one in-house method. Main Outcome Measure(s): The between-kit variability and the effect of the mean (± SD) SHBG level were determined, the results obtained with the use of the kits and the in-house method were compared, and the intraassay variability (i.e., precision) was evaluated. Result(s): Male samples demonstrated a 26.3%-40.8% variance in the results obtained with different kits, which was greatest for samples with the lowest SHBG levels. For female samples, between-kit variability ranged from 57%–115% (average, 77%). The percent deviation of the results obtained with the use of commercial methods from those obtained with the use of our in-house assay was greater for men (mean variance, 194%) than for women (mean variance, 67%). The female pool intraassay coefficient of variation was 3.8% with the use of the in-house method and ranged from 8.9%-21.2% with the use of the commercial kits. The male pool intraassay coefficient of variation was 3.1% with the use of the in-house method and ranged from 3.3%-5.5% with the use of the commercial kits. Conclusion(s): Most commercially available kits for measuring the total serum testosterone level demonstrated significant between-kit variability, which was greatest for female samples. Further, samples with the lowest SHBG levels had the highest between-kit variances. These data strongly suggest that the measurement of total serum testosterone using commercial kits may have limited utility, particularly for the detection of hyperandrogenemia.

Cytomegalovirus infection and viral shedding in the genital tract of infertile couples.

The prevalence of cytomegalovirus (CMV) infection and viral shedding in infertile couples in Taiwan and its role in infertility were studied. Two hundred fifty couples were enrolled in this study. Anti-CMV IgG was measured in serum samples from these infertile couples. Viral shedding examined by DNA hybridization was detected in semen from the husband and cervical mucus from the wife. Anti-CMV IgG was detected in 249 (99.6%) of the 250 male serum samples and in 247 (98.9%) of the 250 female serum samples. Viral shedding was detectable in 83 (33.5%) of 248 semen samples and 83 (33.7%) of 246 cervical mucus samples by dot-blot DNA hybridization assay. Semen quality was not apparently affected by the existence of viral shedding. The co-shedding rate in semen and cervical mucus was high (15.9%). It is concluded that the seroprevalence and genital tract viral shedding were relatively high in infertile couples in Taiwan. Viral shedding did not affect the semen quality. Nevertheless, screening of donor semen is recommended.

Relatively low levels of dimeric inhibin circulate in men and women with polycystic ovarian syndrome using a specific two-site enzyme-linked immunosorbent assay.

The endocrine feedback role of dimeric inhibin on FSH secretion from the pituitary has been well established in many species; however, evidence that inhibin is an important endocrine regulator of FSH in the human is more tenuous. One potential explanation for the equivocal data may be that the inhibin immunoassay used most widely in the human is a heterologous assay with an antiserum that exclusively recognizes the inhibin alpha-subunit in both its monomeric form and in inhibin dimers. The aim of the present study was to quantify serum inhibin levels in a variety of fertile and infertile men and women using a new ultrasensitive enzyme-linked immunosorbent assay that is specific for the dimeric form (alpha/beta) only. The specificity of the present assay was demonstrated by the absence of significant cross-reactivity with Mullerian inhibiting substance, transforming growth factor-beta, activin, FSH, LH, hCG, TSH, and hCG alpha and with the alpha-subunit of inhibin. The assay was sensitive to 1 pg/mL, and serial dilutions of human male and female serum samples paralleled the recombinant 32-kilodalton (kDa) dimeric inhibin standard curve. Complete recovery of exogenous recombinant 32-kDa inhibin added to serum was obtained. Mean serum inhibin levels ranged from a low of 5.7 +/- 0.6 pg/mL in the early follicular phase to 49.0 +/- 11.2 pg/mL in the midluteal phase of the normal menstral cycle and were elevated during ovulation induction (1250 pg/mL) and pregnancy (500 pg/mL). Interestingly, mean levels of dimeric inhibin in women with polycystic ovarian syndrome (PCOS) were indistinguishable from normal follicular phase. The most striking observation was the extremely low mean inhibin levels (< 2 pg/mL) found in normal men, GnRH-deficient men before any pulsatile GnRH treatment, and men with Klinefelter's syndrome, all of which were indistinguishable from levels observed in postmenopausal women. These observations in the male raise the possibly that 1) some forms of circulating and bioactive inhibin in the human are not detected by this assay due to either their conformation or the presence of a unique binding protein for endogenous inhibin that does not bind to recombinant 32-kDa inhibin; or 2) dimeric inhibin is not a major endocrine regulator of FSH in the human male. In addition, the relatively high dimeric inhibin levels at midcycle and in the luteal phase of the normal menstrual cycle, after gonadotropin stimulation, and during pregnancy suggest that dimeric inhibin is predominantly produced by dominant follicles, corpora lutea, and placental tissues.

Blood steroid levels in the goldfish: Measurement of six ovarian steroids in small volumes of serum by reverse-phase high-performance liquid chromatography and radioimmunoassay

A reliable and rapid technique for the measurement of estradiol-17β, testosterone, progesterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one, and cortisol by reverse-phase high-performance liquid chromatography and radioimmunoassay in single female goldfish serum samples of 50 μl was developed. The steroids were extracted with Sep-Pak C18 cartridges after heat treatment. While estradiol-17β was assayed directly after extraction, the other steroids were separated on a μBondapak C18 stainless-steel column with acetonitrile:water (53:47, v/v) in 17 min under isocratic conditions, and then quantitated by specific radioimmunoassays. The technique was validated and was shown to be highly accurate, precise, sensitive, and specific for measuring those particular ovarian steroids in goldfish serum. The steroid levels measured in this way were not significantly different from those measured after separation on a Nova-Pak C18 column with methanol:water (56:44, v/v), where all the individual steroids could be completely resolved during a 40-min run. With this technique, the steroid levels in the serum of the goldfish during the secondary yolk stage, the tertiary yolk stage, and at 0 hr after ovulation were determined. While estradiol-17β was found to be significantly higher in the tertiary yolk stage, testosterone was significantly higher in the secondary yolk stage than in the other two stages. There was no significant difference in the levels of cortisol, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one, and progesterone among the three stages. We conclude that the technique is particularly useful for assaying multiple steroids in very small volumes of biological fluids.

Autoimmune anti-androgen-receptor antibodies in human serum.

Circulating autoantibodies to human and rat androgen receptors are present at high titers in the blood sera of some patients with prostate diseases. The antibodies from some serum samples were associated with a purified IgG fraction and interacted with the 3.8S cytosolic androgen-receptor complexes of rat ventral prostate to form 9- to 12S units. Other serum samples, however, formed 14- to 19S units, suggesting that other immunoglobulins might be involved. In the presence of an anti-human immunoglobulin as a second antibody, the androgen-receptor-antibody complexes could be immunoprecipitated. The antibodies interacted with the nuclear and the cytosolic androgen-receptor complexes, either the DNA-binding or the nonbinding form, but not with receptors for estradiol, progestin, or dexamethasone from a variety of sources. Human testosterone/estradiol-binding globulin, rat epididymal androgen-binding protein, or rat prostate alpha-protein (a nonreceptor steroid-binding protein) also did not interact with the antibodies to form immunoprecipitates. About 37% of male and 3% of female serum samples screened had significant antibody titer. The chance of finding serum with a high titer is much better in males older than 66 years than in the younger males or females at all ages. The presence of the high-titer antibodies may make it possible to prepare monoclonal antibodies to androgen receptors without purification of the receptors for immunization.

Binding of testosterone, 5α-dihydrotestosterone and 5α-androstane(3α- and 3β),17β-diols to serum proteins in the rat

Experiments with polyacrylamide gel electrophoresis at steady state conditions, indicated that testosterone, 5α-dihydrotestosterone, 3α-androstanediol and 3β-androstanediol bound only to albumin in rat serum. The extent of binding of several C 19-steroids to serum proteins was measured in rat blood utilizing equilibrium dialysis. The values for the adult male rat were as follows (mean ± SEM): testosterone 78.3 ± 0.4%, 5α-dihydrotestosterone 81.8 ± 0.4%, 3α-androstanediol 95.5 ± 0.1%; 3β-androstanediol 95.2 ± 0.1%. The values for the latter two are significantly higher than those for the first two which in turn differ significantly from each other. The values for adult female and castrated male serum samples were not significantly different from those of the normal male. The results obtained for the extent of binding of C 19-steroids in a 5-g% albumin solution (which is similar to male rat serum albumin concentration) did not differ significantly from values for serum indicating that the binding in the latter is due primarily to albumin. The K A ,s for C 19-steroid binding to albumin were: testosterone 2.22 × 10 4 l/mol; 5α-dihydrotestosterone 3.46 × 10 4 l/mol; 3α-androstanediol 1.36 × 10 5 l/mol and 3β-androstanediol 1.20 × 10 5 l/mol. When correlated to our previously reported metabolic clearance rates (MCR) it was concluded that binding to serum proteins does not account for the significant difference between the MCR for testosterone and 5α-dihydrotestosterone but may account for the lower MCR's for the 3α- and 3β-androstanediols.

Human prostatic acid phosphatases: III. Counterimmunoelectrophoresis for rapid identification.

When serial dilution of standard prostatic acid phosphatases (PAP) was reacted with constant amounts of anti-PAP serum by counterimmunoelectrophoresis (CIEP), the detection end point of enzyme concentration was 0.25 ng in a 10 microliter sample volume. The PAP concentrations in unknowns can be quantitated by comparing the dilution end points of reference PAP with the testing samples. Serum PAP levels were determined by a radioimmunoassay (RIA) and CIEP using normal male and female sera and serum samples from patients with prostatic cancer and nonprostatic tumors. An excellent correlation was observed between the two assay results. According to RIA data, the concentration of PAP higher than 0.4 ng per 10 microliter (or 4.0 ng per 100 microliter) signify the elevation of serum or bone marrow PAP level beyond normal range (normal value 1.6 +/- 0.8 ng/100 microliter). Thus, the CIEP assay will be a simple and reliable screening method for the serum PAP levels in the clinical diagnosis of prostatic cancer.