Correction to: EStradiol and PRogesterone in In vitro ferTilization (ESPRIT): a multicenter study evaluating third­ versus second­generation estradiol and progesterone immunoassays.

The article "EStradiol and PRogesterone in In vitro ferTilization (ESPRIT): a multicenter study evaluating third­ versus second­generation estradiol and progesterone immunoassays.

EStradiol and PRogesterone in In vitro ferTilization (ESPRIT): a multicenter study evaluating third- versus second-generation estradiol and progesterone immunoassays.

PURPOSE: To assess estradiol (E2) and progesterone levels during ovarian stimulation determined by third-generation (Gen III) and second-generation (Gen II) Elecsys® immunoassays. METHODS: E2 and progesterone concentrations were measured using Elecsys® Gen III and Gen II immunoassays, and progesterone concentrations on the day of ovulation triggering were determined by LC-MS/MS. This was a retrospective, non-interventional study conducted at European tertiary referral infertility clinics in women aged 18-45 years, with a body mass index 18-35 kg/m2, regular menses, and both ovaries. RESULTS: Serum samples were obtained from 230 women classified by oocyte retrieval as poor (33.0%; 0-3 oocytes), normal (40.9%; 4-15 oocytes), or high (26.1%; > 15 oocytes) responders. E2 and progesterone levels increased during ovarian stimulation, with greatest increases observed in high responders. Elecsys® Gen III and Gen II assay results were highly correlated for E2 (Pearson's r = 0.99) and progesterone (r = 0.89); Gen III results were lower than Gen II for both E2 and progesterone. On the day of triggering, Gen III E2 and progesterone levels showed a difference of - 15.0% and - 27.9%, respectively. Progesterone levels (on day of triggering) measured by LC-MS/MS correlated better with Gen III (0.98) than Gen II (0.90). Mean relative differences for Gen III and Gen II assays versus LC-MS/MS were 14.6% and 62.8%, respectively. CONCLUSION: E2 and progesterone levels determined with Elecsys® Gen II and III assays were highly correlated; results were lower for Gen III versus Gen II. Differences observed for progesterone on the day of triggering may be clinically relevant.

Multicenter analytical performance evaluation of a fully automated anti-Müllerian hormone assay and reference interval determination.

OBJECTIVES: Anti-Müllenria hormone (AMH) is an established biomarker for assessing ovarian reserve and predicting response to controlled ovarian stimulation. Its routine clinical use is hampered by the variability and low-throughput of available enzyme-linked immunosorbent assays (ELISA). The presented study examined if a fully automated AMH electrochemiluminescence assay (ECLIA; Elecsys® AMH assay, Roche Diagnostics) was suitable for measuring AMH levels in healthy women and in those diagnosed with polycystic ovary syndrome (PCOS). DESIGN AND METHODS: Five European laboratories evaluated the Elecsys® AMH assay independently under routine conditions over eight months. Within-run imprecision, repeatability, intermediate precision, linearity and functional sensitivity were assessed. The Elecsys® AMH assay was compared to a manual ELISA microtiter plate format test (AMH Gen II ELISA, modified version; Beckman Coulter Inc.) using 1729 routine serum samples. AMH reference intervals were determined in 887 healthy women with regular menstrual cycle aged 20­50 years, and 149 women diagnosed with PCOS. RESULTS: The fully automated Elecsys® AMH assay showed excellent precision, linearity, and functional sensitivity. The coefficient of variation was 1.8% for repeatability and 4.4% for intermediate precision. Values measured with the Elecsys® AMH assay were highly correlated with the manual ELISA method (modified version) but 24­28% lower. Reference intervals showed the expected AMH decline with age in healthy women and increased AMH levels in women with PCOS. CONCLUSIONS: The Elecsys® AMH assay demonstrated good precision under routine conditions, and is suitable for determining AMH levels in serum and lithium-heparin plasma.

One left dorsolateral prefrontal cortical HF-rTMS session attenuates HPA-system sensitivity to critical feedback in healthy females.

OBJECTIVE: Although left dorsolateral prefrontal cortical (DLPFC) repetitive Transcranial Magnetic Stimulation (rTMS) is used to treat major depression, its underlying neurophysiological working mechanism remains to be determined. Prior research suggested that the clinical effects could be mediated by affecting the hypothalamic-pituitary-adrenal (HPA) system, but experimental studies in healthy individuals did not yield clear results. However, in healthy individuals, the influence of HF-rTMS on the HPA-system may only be detected when it is challenged. METHODS: In 30 rTMS naïve healthy females we evaluated the effect of one sham-controlled high frequency (HF)-rTMS session applied to the left DLPFC on the stress hormone cortisol by collecting salivary cortisol samples. In order to increase stress levels, 5min after stimulation, all participants performed the Critical Feedback Task (CFT), during which they were criticized on their performance. To take possible mood influences into account, all participants were also assessed with Visual Analogue Scales (VAS). RESULTS: The experimental procedure did not affect mood differently in the real or sham stimulation. Area under the curve (AUCi) analysis showed that one real HF-rTMS session significantly influenced HPA-system sensitivity, as demonstrated by a decrease in cortisol concentrations. The sham procedure yielded no effects. CONCLUSIONS: In line with former observations in major depression, one real left DLPFC HF-rTMS session significantly influenced HPA-system sensitivity in experimentally stressed females, resulting in decreases in cortisol levels.

Gender-specific alterations in salivary cortisol levels in pubertal intracytoplasmic sperm injection offspring.

BACKGROUND: There is accumulating evidence that in vitro conception in humans may be associated with adverse health outcomes later in life. It has been proposed that suboptimal early life conditions may 'program' key endocrine systems. A disturbance of the hypothalamic-pituitary-adrenal (HPA) axis leading to alterations in cortisol secretion in the offspring may be such a mechanism. To date, no data on cortisol levels in children conceived by intracytoplasmic sperm injection (ICSI) are available in the literature. METHODS: In this cross-sectional study, salivary cortisol known as a key regulator of metabolism was measured and results were compared between 201 pubertal ICSI children and 196 spontaneously conceived (SC) counterparts. RESULTS: ICSI females had lower mean salivary cortisol levels (9.0 µg/l; 95% CI 8.1-9.9) than SC females (10.6 µg/l; 95% CI 9.7-11.5; p = 0.01). This difference remained after adjusting for current characteristics, early life factors and maternal characteristics. In ICSI males, no difference in cortisol levels was found in comparison with the SC group. CONCLUSION: In our study, 14-year-old female but not male ICSI teenagers were found to have lower salivary cortisol concentrations in comparison with SC peers. However, before definite conclusions can be drawn, our results should be completed by longitudinal sampling.

Salivary testosterone concentrations in pubertal ICSI boys compared with spontaneously conceived boys.

BACKGROUND To date, no data exist about Leydig cell function of pubertal boys born after ICSI. To evaluate a potential risk of gonadal dysfunction in children born from fathers with compromised fertility, testicular function was assessed by the measurement of salivary testosterone. METHODS Morning salivary testosterone levels at the age of 14 years were compared between 58 ICSI teenagers who are part of the oldest ICSI cohort, and 62 boys born after spontaneous conception (SC). RESULTS Salivary testosterone levels were comparable between ICSI (113 ± 42 pg/ml) and SC (123 ± 56 pg/ml) teenagers at the age of 14 years. In the ICSI group, testosterone levels in boys from fathers with severe oligozoospermia were not different from concentrations in boys from fathers without severe oligozoospermia (115.5 ± 43 and 109 ± 41 pg/ml, respectively). CONCLUSIONS At the age of 14 years, pubertal ICSI boys show testosterone levels comparable to their peers born after SC. ICSI adolescents fathered from men with severely compromised spermatogenesis show testosterone levels comparable to those from fathers with normal spermatogenesis. This notwithstanding, further follow-up of ICSI teenagers into adulthood is mandatory to confirm a normal gonadal function.

Serum inhibin B concentrations in pubertal boys conceived by ICSI: first results.

BACKGROUND: Currently, no published data exist about the gonadal function of children born after ICSI. To evaluate potential risk of testicular seminal dysfunction in boys born to fathers with compromised spermatogenesis, serum inhibin B (as a marker for spermatogenesis) was assessed. METHODS: We recruited 50 pubertal adolescents from the oldest cohort of infants born following ICSI. Cross-sectional serum inhibin B levels of all 50 ICSI adolescents, and longitudinal serum inhibin B (assessed at 8 and 14 years) in 25 boys, are reported. RESULTS: A statistically significant increase in inhibin B levels was observed between 8 (mean 69 ng/l, SD ± 35) and 14 years (mean 145 ng/l, SD ± 41; P < 0.001). In three quarters of the ICSI boys an increase in serum inhibin B levels of at least 30% between 8 and 14 years was observed. In all but 4 of the 14-year-old ICSI boys serum inhibin B was normal. Serum inhibin B levels in boys from fathers with severe oligozoospermia did not differ from concentrations in boys from fathers without severe oligozoospermia (154 ± 51 and 142 ± 47 ng/l, respectively; P = 0.4). CONCLUSIONS: The majority of ICSI boys have a significant increase in serum inhibin B, attaining normal values for pubertal status at the age of 14 years. ICSI adolescents from fathers with severely compromised spermatogenesis do not have lower inhibin B levels than those with fathers with normal spermatograms. Further follow-up of the spermatogenic potential of ICSI teenagers up to young adulthood is mandatory to confirm a normal reproductive capacity.

Multicenter evaluation of the first automated Elecsys sFlt-1 and PlGF assays in normal pregnancies and preeclampsia.

OBJECTIVES: Performance evaluation of Elecsys sFlt-1 and PlGF assays. DESIGN AND METHODS: Within-, between-run, total imprecision, functional sensitivity, inter-laboratory comparison, method comparison and lot-to-lot reproducibility were evaluated. RESULTS: Within- and between-run CVs were below 4% for sFlt-1 >60 and PlGF > 20 pg/mL. Total imprecision CVs were below 4.3%. Functional sensitivity was < 5 pg/mL. Inter-laboratory CVs were <5%. Elecsys correlated well with Quantikine VEGF-R1 (r=0.960) and PlGF (r=0.968). Lot-to-lot comparisons yielded highly correlated results (r>0.999). In healthy pregnancies, the median levels of sFlt-1 remained constant in first (1107 pg/mL) and second trimesters (1437 pg/mL) but increased in the third trimester (2395 pg/mL), while median PlGF levels increased in the first (30 pg/mL) and second trimesters (279 pg/mL) and peaked at 29 to 32 weeks (626 pg/mL) and decreased thereafter (340 pg/mL). The sFlt-1/PlGF ratio is highest in the first trimester (median: 28) but remained constant in the second (median: 4.7) and third trimesters (median: 5.1). In PE/HELPP samples matched for gestational age the sFlt-1 levels were significantly higher (6894-34,624 pg/mL), whereas PlGF levels were lower (9.2-80 pg/mL) and the median sFlt-1/PlGF ratio is much higher (461; range: 121-2614) than in apparently healthy pregnancies (3.6; range: 0.3-105). CONCLUSION: The new Roche Elecsys sFlt-1 and PlGF immunoassay showed excellent precision and reliability. There was a clear difference in the Elecsys sFlt-1/PlGF ratio between samples obtained from women with apparently normal pregnancy at the time of blood collection and those diagnosed with PE/HELLP at the same age of gestation.

The impact of one HF-rTMS session on mood and salivary cortisol in treatment resistant unipolar melancholic depressed patients.

BACKGROUND: Recent studies indicate that medication resistant depressed patients can be successfully treated by a series of sessions of High Frequency repetitive Transcranial Magnetic Stimulation (HF-rTMS), delivered on the left dorsolateral prefrontal cortex (DLPFC). However, changes in subjectively experienced mood give only limited insight into the underlying physiological responses. Previous studies in depressed patients, as well as in healthy volunteers, have reported a possible impact of HF-rTMS on the hypothalamic-pituitary-adrenal (HPA) axis. OBJECTIVE: We wanted to evaluate the emotional and neurobiological impact of one session of HF-rTMS applied on the left DLPFC in a sample of unipolar treatment resistant depressed patients of the melancholic subtype. METHODS: 20 right-handed antidepressant-free depressed patients were studied using a sham-controlled, 'single' blind, crossover design. We examined subjective mood changes with Visual Analogue Scales (VAS). To examine HF-rTMS effects on the HPA-axis, we analyzed salivary cortisol levels. Mood assessment and salivary cortisol levels were assessed before and immediately after stimulation. To detect any delayed effects, all measurements were also re-assessed 30 min post HF-rTMS. The left DLPFC was determined under MRI guidance. RESULTS: One session of HF-rTMS did not result in any subjectively experienced mood changes. However, salivary cortisol concentrations decreased significantly immediately and 30 min after active HF-rTMS. CONCLUSIONS: Although one session of HF-rTMS on the left DLPFC did not influence mood subjectively in melancholic unipolar depressed patients, we found support for the hypothesis that a single session has a significant impact on the HPA-axis, as measured by salivary cortisol. Our results may provide more insight into the underlying working mechanisms of HF-rTMS in unipolar melancholic depression, and could add further information about endocrinological functioning in affective disorders.

Thyroid autoimmunity and female infertility.

In infertile women, the prevalence of thyroid autoimmunity (TAI) is significantly higher compared to that in parous age-matched women. This is especially the case in women with endometriosis and the polycystic ovarian syndrome. TAI does not interfere with normal fetal implantation and comparable pregnancy rates have been observed after assisted reproductive technology (ART) in women with and without TAI. During the first trimester however, pregnant women with TAI carry a significantly increased risk for a miscarriage compared to women without TAI, even when euthyroidism was present before pregnancy. It has further been demonstrated that controlled ovarian hyperstimulation (COH) in preparation for ART has a significant impact on thyroid function, particularly in women with TAI. It is therefore advised to measure thyroid function and detect TAI in infertile women, before ART, and to follow-up these parameters after COH and during pregnancy when TAI was initially present. Women with thyroid dysfunction before or at early gestation stages should be treated with 1-thyroxine to avoid assisted pregnancy or further pregnancy complications. Whether thyroid hormones should be given prior to or during pregnancy in euthyroid women with TAI remains controversial and needs further investigation.

Variability among five different commercial IGF-1 immunoassays in conditions of childhood-onset GH deficiency and GH therapy.

IGF-1 measurement is used for screening of GH deficiency and monitoring of GH therapy in children. However, several commercial immunoassays are currently used and reference values provided by manufacturers are very different. The aim of this study was to compare commercial IGF-1 assays 1) in terms of absolute values and 2) in terms of clinical interpretation of results based on IGF-1 reference values in serum samples from children with GH therapy, with untreated GH deficiency and with obesity. Serum samples of 9 patients were sent frozen to 5 university hospitals using 5 different IGF-1 assays. The inter-laboratory coefficient of variation (CV) was calculated for the 9 samples. For clinical interpretation, results were expressed as SD scores based on reference values provided by manufacturers (and used in these laboratories). The mean inter-laboratory CV (range) for the 9 serum samples was 25.8% (16.7-35.9%). Major variability was noted in the SD-scores between IGF-1 assays for 3 tested serum samples from GH-treated patients with a difference between the lowest and highest SD score of 2.6 up to 3.2. In conclusion, there is a large variability among commercial IGF-1 immunoassays, not only in terms of absolute values, but also in terms of clinical interpretation in pediatric serum samples. There is a need for IGF-1 immunoassay harmonization and for the establishment of adequate reference values.

Macroprolactinemia: clinical significance and characterization of the condition.

The purpose of this study was to characterize the clinical picture of macroprolactinemic patients and to further assess whether macroprolactinemia was part of an auto-immune syndrome. Eighty-two hyperprolactinemic (serum PRL > 1000 mU/l) patients were investigated and the PEG precipitation test identified 14 patients with macroprolactinemia (bb PRL). They were submitted to a hormonal and autoimmune screening and an IV TRH test. Bioactivity of their serum prolactin was evaluated, using an Nb2 assay. The biochemical nature of bb-PRL was investigated by immunoprecipitation with anti-IgG antibodies. Seventy-nine percent of the studied patients presented with infertility, amenorrhoea, galactorrhoea, mastodynia, gynaecomastia or erectile dysfunction. In most cases, however, these symptoms could be explained by the presence of other non hyperprolactinemia-related pathology. Despite the finding of in vitro biological activity in all macroprolactinemic sera tested, our results suggest a variable in vivo bioactivity of bb-PRL, probably related to a reduced capacity to cross vascular endothelium. In this study, we demonstrated that in 12 out of 13 samples (85%), bb-PRL consisted of PRL-IgG complexes. There was no clinical or laboratory evidence of auto-immunity.

Profound LH suppression after GnRH antagonist administration is associated with a significantly higher ongoing pregnancy rate in IVF.

BACKGROUND: The significance of suppressed LH levels in GnRH antagonist cycles for IVF outcome is currently unknown. The purpose of this study was to evaluate prospectively the association between LH levels and ongoing pregnancy achievement after GnRH antagonist initiation in IVF cycles. METHODS: Ovarian stimulation with a fixed dose of 200 IU recombinant FSH and daily GnRH antagonist (ganirelix) 0.25 mg from day 6 of stimulation was initiated in 116 women. Patients were not pretreated with an oral contraceptive. Induction of final oocyte maturation was performed with HCG 10,000 IU as soon as three follicles of > or =17 mm were present in ultrasound, and was followed by oocyte pick-up, conventional IVF or ICSI, and embryo transfer. The luteal phase was supplemented with vaginal progesterone. RESULTS: A significant decrease of both ongoing pregnancy rate and implantation rate was present across groups of patients with increasing LH levels. The highest implantation rate and ongoing pregnancy rate was present in those patients with LH levels on day 8 of stimulation < or =0.5 IU/l. CONCLUSIONS: Profound suppression of LH on day 8 of stimulation is associated with a significantly higher chance of achieving an ongoing pregnancy. More studies are necessary to evaluate this phenomenon further.

Serum inhibin B cannot predict testicular sperm retrieval in patients with non-obstructive azoospermia.

BACKGROUND: Serum inhibin B, a direct product of the Sertoli cells, may serve as a marker of spermatogenesis. The present retrospective study aimed at evaluating the predictive value of inhibin B for retrieving testicular sperm in non-obstructive azoospermic men. METHODS: The serum inhibin B concentration before sperm retrieval was reviewed in 185 non-obstructive azoospermic patients. RESULTS: Testicular sperm were successfully recovered in 92 of 185 patients (49.7%). The mean inhibin B concentration in these patients was 37.3 pg/ml. No sperm were found in 93 patients (50.3%), and the mean serum inhibin B concentration was 44.9 pg/ml. The discrimination between successful and unsuccessful sperm retrieval was analysed using the receiver operating characteristics (ROC) curve analysis. The best discriminating inhibin B concentration was 13.7 pg/ml (sensitivity 44.6%, specificity 63.4%) with an area under the ROC curve (AUC) of 0.51. Combining both serum FSH and inhibin B did not improve the predictive value: the AUC of inhibin B in men with a serum FSH concentration <25 and > or = 25 IU/l (being the best threshold value in the population studied) was respectively 0.53 and 0.50. The AUC of the inhibin B:FSH ratios was 0.55. CONCLUSIONS: This analysis shows that inhibin B, either alone or in combination with serum FSH, fails to predict the presence of sperm in men with non-obstructive azoospermia undergoing testicular sperm extraction.

Rapid detection of macroprolactin in the form of prolactin-immunoglobulin G complexes by immunoprecipitation with anti-human IgG-agarose.

Immunoprecipitation with anti-human immunoglobulin G-agarose was evaluated for the detection of prolactin-immunoglobulin G (PRL-IgG) complexes in macroprolactinemic samples from hyperprolactinemic patients (prolactin measured using the automated Elecsys PRL assay; Roche Diagnostics, Mannheim, Germany). Using the polyethylene glycol (PEG) precipitation test on serum samples with PRL above 1000 mIU/l, we detected macroprolactin in 38 out of 175 samples with a recovery below 50%. Gel filtration chromatography on a subset of hyperprolactinemic samples confirmed that macroprolactin was the predominant immunoreactive form of PRL in samples which showed a recovery < or = 50%. In 37 out of 38 samples containing macroprolactin, immunoprecipitation with anti-human IgG (anti-hIgG)-agarose revealed a higher PRL-binding (19.1-91.3%) than in the sera containing monomeric PRL (< 10%). In conclusion, we found that PEG precipitation detected macroprolactin in 21.7% of samples with elevated PRL levels as measured by the Elecsys PRL assay, and that in the vast majority of these samples the presence of PRL-IgG complexes could be demonstrated by immunoprecipitation with anti-hIgG-agarose. In view of the high prevalence of PRL-IgG complexes in hyperprolactinemic patients and the simplicity of the test, immunoprecipitation with anti-hIgG-agarose might play a role in the routine laboratory handling of hyperprolactinemic samples, especially with regards to PRL assays where PEG causes interference.

Oocyte and embryo quality as well as pregnancy rate in intracytoplasmic sperm injection are not affected by high follicular phase serum progesterone.

The effect of elevated serum progesterone concentrations (> 1 ng/l) on or before the day of human chorionic gonadotrophin (HCG) injection on the outcome of women receiving gonadotrophin-releasing hormone analogue (GnRHa)/human menopausal gonadotrophin (HMG) for ovarian stimulation prior to intracytoplasmic sperm injection (ICSI) was evaluated. A total of 1275 ICSI cycles were analysed retrospectively. In 53 cycles (4.5%), serum progesterone concentrations were > 1 ng/ml. Patients in the high progesterone group had significantly higher oestradiol and luteinizing hormone concentrations on the day of HCG injection. The characteristics of the cumulus-corona cell complexes and the nuclear maturity of the oocytes were similar in the groups of patients with high and low serum progesterone levels. Fertilization and cleavage rates as well as embryo quality were not different in the two groups. No difference in implantation or clinical pregnancy rates was observed between the high progesterone and low progesterone groups. Moreover, the cumulative exposure to progesterone during the follicular phase, as expressed by the area under the curve (AUC), and the duration of exposure to high serum progesterone levels (> 1 ng/ml) were not significantly different between pregnant and non-pregnant women in the high progesterone group. We conclude that in ICSI cycles pretreated with GnRHa, increased serum progesterone concentrations on or before the day of HCG injection do not affect ICSI outcome.

Positive interference of merthiolate in the TDx digoxin assay in control samples used in the Belgian External Quality Assessment (EQA) Scheme.

We investigated the possible origin of the spuriously high results observed with the Abbott TDx Immunoassay in the 1991 Belgian external quality assessment scheme for digoxin. The present work ascribes this discrepancy to a matrix effect induced by the addition of merthiolate as preservative to the control samples. It consequently stresses the importance of avoiding the use of this compound for preparing such samples.