STUDY QUESTION How do anti-Müllerian hormone (AMH) serum concentrations and follicle densities (FDs) change with age and disease and what are the implications for fertility preservation? SUMMARY ANSWER AMH concentrations and FD do not correlate in young women, and AMH but not FD is reduced in some diseases, limiting the value of AMH as a predictive parameter of ovarian tissue transplantation. WHAT IS KNOWN ALREADY AMH is widely used as a parameter to estimate the ovarian reserve. However, the reliability of AMH to predict total number of follicles and the FD is questionable. Women with lymphoma and leukaemia have been shown to have reduced AMH concentrations, but it is unknown if the FD is also reduced. In fertility preservation it is essential to estimate the correct total number of follicles and the FD, as ovarian tissue should only be cryopreserved if ovarian reserve is high. Furthermore, the amount of tissue to be transplanted should be based on the estimation of the real FD. STUDY DESIGN, SIZE, DURATION This retrospective observational study included 830 women (mean ± SD age, 28.2 ± 6.81 years; range, 4–43 years) with malignant (n = 806) and benign (n = 24) diseases who cryopreserved tissue in a single centre as part of a national fertility preservation programme. Females with ovarian surgery or known predispositions for a reduced ovarian reserve were excluded. AMH concentrations and FD were evaluated from March 2011 to September 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS AMH concentrations were analysed before gonadotoxic therapies. Standardized biopsies, obtained from different areas of ovarian cortex, were collected. FD was analysed after tissue digestion and calcein staining and was expressed as average number of primordial and primary follicles count per 3 mm biopsy and per cubic millimeter tissue. AMH concentrations and FD were analysed in relation to age and diagnosis group. Both parameters were age adjusted, and associations between the different diagnosis groups and AMH versus FD were assessed. MAIN RESULTS AND THE ROLE OF CHANCE Mean ± SD AMH concentration was 3.1 ± 2.81 g/ml, mean FD per 3 mm biopsy was 137 ± 173.9 and 19.4 ± 24.60 per mm3. Maximum AMH concentrations were found in children and teenagers at the age of 6–10 years (5.71 ng/ml) and in adults at the age of 21–25 years (3.33 ng/ml). FD was highest in young children up to an age of 15 years and decreased with increasing age. AMH and FD were not correlated in women ≤20 years and weakly to moderately correlated in women 21–40 years (r = 0.24–0.39). Age-adjusted correlations between AMH and FD were demonstrated in several diagnosis groups such as breast cancer, leukaemia, sarcoma, gastrointestinal cancer and gynaecological cancer but not in the groups exhibiting Hodgkin’s and non-Hodgkin’s lymphoma, cerebral cancer, other types of malignancies and other types of benign diseases. Further statistical analysis supported the finding that, in some diagnosis groups such as Hodgkin’s lymphoma and in gynaecological cancer, AMH concentrations but not FDs are reduced, questioning the prognostic accuracy of AMH for the FD in these diseases. LIMITATIONS, REASONS FOR CAUTION Even though biopsies were taken from different sites, heterogenous distribution of follicles might have had some effect on the accuracy of the analysis. WIDER IMPLICATION OF THE FINDINGS AMH should be used with care to estimate the total ovarian reserve and FD of cancer patients in young women in some diseases. Therefore, calculating the amount of ovarian tissue to be transplanted based solely on AMH might be inaccurate whereas FD might be a better parameter. STUDY FUNDING/COMPETING INTEREST(S) The study did not receive any exterior funding.

STUDY QUESTION: How do anti-Mullerian hormone (AMH) serum concentrations and follicle densities (FDs) change with age and disease and what are the implications for fertility preservation? SUMMARY ANSWER: AMH concentrations and FD do not correlate in young women, and AMH but not FD is reduced in some diseases, limiting the value of AMH as a predictive parameter of ovarian tissue transplantation. WHAT IS KNOWN ALREADY: AMH is widely used as a parameter to estimate the ovarian reserve. However, the reliability of AMH to predict total number of follicles and the FD is questionable. Women with lymphoma and leukaemia have been shown to have reduced AMH concentrations, but it is unknown if the FD is also reduced. In fertility preservation it is essential to estimate the correct total number of follicles and the FD, as ovarian tissue should only be cryopreserved if ovarian reserve is high. Furthermore, the amount of tissue to be transplanted should be based on the estimation of the real FD. STUDY DESIGN, SIZE, DURATION: This retrospective observational study included 830 women (meanSD age, 28.26.81 years; range, 4-43 years) with malignant (n = 806) and benign (n = 24) diseases who cryopreserved tissue in a single centre as part of a national fertility preservation programme. Females with ovarian surgery or known predispositions for a reduced ovarian reserve were excluded. AMH concentrations and FD were evaluated from March 2011 to September 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: AMH concentrations were analysed before gonadotoxic therapies. Standardized biopsies, obtained from different areas of ovarian cortex, were collected. FD was analysed after tissue digestion and calcein staining and was expressed as average number of primordial and primary follicles count per 3 mm biopsy and per cubic millimeter tissue. AMH concentrations and FD were analysed in relation to age and diagnosis group. Both parameters were age adjusted, and associations between the different diagnosis groups and AMH versus FD were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: MeanSD AMH concentration was 3.1 +/- 2.81 g/ml, mean FD per 3 mm biopsy was 137 +/- 173.9 and 19.4 +/- 24.60 per mm(3). Maximum AMH concentrations were found in children and teenagers at the age of 6-10 years (5.71 ng/ml) and in adults at the age of 21-25 years (3.33 ng/ml). FD was highest in young children up to an age of 15 years and decreased with increasing age. AMH and FD were not correlated in women <= 20 years and weakly to moderately correlated in women 21-40 years (r =0.24-0.39). Age-adjusted correlations between AMH and FD were demonstrated in several diagnosis groups such as breast cancer, leukaemia, sarcoma, gastrointestinal cancer and gynaecological cancer but not in the groups exhibiting Hodgkin's and non-Hodgkin's lymphoma, cerebral cancer, other types of malignancies and other types of benign diseases. Further statistical analysis supported the finding that, in some diagnosis groups such as Hodgkin's lymphoma and in gynaecological cancer, AMH concentrations but not FDs are reduced, questioning the prognostic accuracy of AMH for the FD in these diseases. LIMITATIONS, REASONS FOR CAUTION: Even though biopsies were taken from different sites, heterogenous distribution of follicles might have had some effect on the accuracy of the analysis. WIDER IMPLICATION OF THE FINDINGS: AMH should be used with care to estimate the total ovarian reserve and FD of cancer patients in young women in some diseases. Therefore, calculating the amount of ovarian tissue to be transplanted based solely on AMH might be inaccurate whereas FD might be a better parameter.

Objectives: To evaluate the prognostic value of anti-Mullerian hormone (AMH) levels in estimating the ovarian density of primordial and primary follicles, which can be assumed to reflect the real ovarian reserve. Study design: A total of 537 women, average age 30.4 years (range 8.0-43.7 years), underwent ovarian tissue cryopreservation prior to gonadotoxic therapies due to malignant diseases which do not affect ovarian reserve parameters. Standardized ovarian biopsies were obtained, and follicular density was analysed. The prognostic accuracy of serum AMH in estimating ovarian follicle density was evaluated. Main outcome measures: Histologically determined follicle density, AMH serum concentration and their correlation. Results: In children, follicle density was high but AMH concentration was low. AMH concentration was predicted to be maximum at the age of 15.5 years. In women aged over 15.5 years, the relationship between AMH concentration and follicle density was evaluated. Crude analysis revealed that serum AMH levels and follicular density were moderately correlated (r = 0.34, p < 0.001). From the adjusted regression model the predicted value of follicle density of women aged 20, 30 and 40 years as well as the associated 50 % and 95 % prediction intervals (50 % PI and 95 % PI, respectively) were calculated. For example, for women aged 40 years with a serum AMH level of 1 ng/ml, a follicle density of 2.3/mm(3) (50 %PI: [1.1, 4.6]; 95 %PI: [0.3, 18]) was predicted. These large prediction intervals demonstrate the low predictive value of serum AMH for the ovarian follicle density. Conclusions: Serum AMH levels have limited prognostic value for the follicle density and therefore for the real ovarian reserve.

Objective Nuchal translucency (NT) measurement in assisted reproduction treatment (ART) twins is less extensively investigated. Therefore, the present study compared NT measurements of spontaneously conceived twins with ART twins in dichorionic (DC) and monochorionic (MC) pregnancies.Methods Retrospective analysis of 706 unaffected twins between 11 + 0 and 13 + 6 weeks conceived either spontaneously (n=362) or with ART (n=344). The group with spontaneous conception included 234 DC (64.6%) and 128 MC (35.4%) pregnancies. In the ART group, 326 were DC (94.7%) and 18 were MC (5.3%). NT values were transformed into multiples of median (MoM).Results In the DC group, no significant differences between ART and spontaneously conceived twins (NT MoM 1.06 +/- 0.28 vs 1.03 +/- 0.29; p > 0.05) were observed. NT MoM of MC ART twins was higher compared with spontaneous MC twins (1.23 +/- 0.82 vs 0.99 +/- 0.27; p = 0.011). Although the incidence of twin-to-twin transfusion syndrome (TTTS) was higher among ART twins (11.1% vs 4.7%), inter-twin NT difference was similar between pregnancies with TTTS (0.42 +/- 0.21) or without (0.40 +/- 0.71).Conclusions Measurement of NT thickness in DC ART twins achieves comparable results with twins conceived spontaneously. Conclusions in MC twins are limited; however, higher NT MoM was not related to TTTS or selective intrauterine growth restriction. (C) 2013 John Wiley & Sons, Ltd.

Introduction Human milk (HM) contains antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase. Melatonin (MT) shows antioxidative properties both directly by stimulating the activities of antioxidant enzymes and indirectly by scavenging free radicals. The aim of this study was to evaluate the effect of MT status on the level of SOD, GSH-Px 3 and the total antioxidant capacity (TAC) of HM. Material and Methods 114 samples of HM were collected from 22 healthy women during daytime (DT; 10a.m.-9.59p.m.) and nighttime (NT; 10p.m.-9.59a.m.). MT, SOD, GSH-Px3 levels and TAC were assayed and compared between DT-HM and NT-HM. Moreover, the Pearson’s correlation coefficient was calculated between MT and SOD, GSH-Px3 and TAC. Results MT showed a circadian rhythm with high levels at NT and low levels at DT (mean± SEM; 9.2±1.2 vs. 2.0±0.3 pg/ml, respectively, p<0.0001), while SOD, GSH-Px3 and TAC had no circadian changes in HM (p>0.05). There was no correlation between MT levels and SOD, GSH-Px3 levels and TAC. Conclusion The MT status influence neither SOD and GSH-Px3 levels nor TAC of HM. It should be pointed that our results represent only the effect of MT on the extracellular component of the antioxidant system and it does not exclude the possibility that high MT concentrations in HM may induce the intracellular and mitochondrial antioxidant enzyme system in the gastrointestinal tract.

Die Messung der Nackentransparenz (NT) ist eine effektive und validierte Methode um Aneuploidien zu entdecken (1). Einige Studien zeigten eine erhöhte NT bei nach Methoden der assistierten Reproduktion (ART) konzipierten Einlingen, es fehlen jedoch Studien zur NT-Messung bei Zwillingsschwangerschaften nach ART, welche die unterschiedliche Chorionizität berücksichtigen (2). Die vorliegende Studie vergleicht (i) die NT spontan konzipierter Zwillinge mit denen nach ART, und (ii) vergleicht die NT dichorialer (DC) und monochorialer (MC) Zwillinge der verschiedenen Gruppen.

Introduction: Considering the potential immunomodulatory role of melatonin and its direct antioxidant activity, disturbances of the melatonin secretion pattern in the septic conditions could be particularly unfavorable. The aim of this study was to evaluate the nocturnal melatonin concentration (NMC) in children with sepsis at the pediatric intensive care unit (PICU). Material and Methods: This observational study involved 15 septic and 14 non-septic patients at the PICU, University of Cukurova, Adana, Turkey. An eye mask was used between 8pm and 8am in all patients. Blood samples for the determination of NMC were obtained from each patient at 3 am on day 1, 2, 3, 5 and 10. Results: The median NMCs were not significantly different between septic and non-septic patients: Median(IQR), 89.6pg/ml (39.1-168.9pg/ml) and 61.3pg/ml (28.5-133.5pg/ml); 102.2pg/ml (50.0- 441.2pg/ml) and 59.0pg/ml (16.4-293.0pg/ml); 73.8pg/ml (39.7-406.2pg/ml) and 97.0pg/ml (26.5- 154.2pg/ml); 78.1pg/ml (38.6-179.3pg/ml) and 153.4pg/ml (45.5-231.1pg/ml); 86.8pg/ml (21.1- 137.5pg/ml) and 120.6pg/ml (17.0-208.3pg/ml) at day 1, 2, 3, 5 and 10, respectively (p>0.05). The septic patients showed no significant changes of NMCs between day1 and each other study day (p>0.05). Presence of catecholamine, sedation and mechanical ventilation had no effect on the NMCs in septic patients (p>0.05). There was no correlation between severity of disease (PRISM-II-Score) and NMC on day 1 (p>0.05). Conclusion: The present study showed that the NMC is not affected in septic PICU patients despite severe disease and it is independent of the treatment. Further investigations are needed to identify any additional benefits of the treatment with melatonin in PICU patients with sepsis.

First-trimester serum markers in 110 in-vitro fertilization (IVF) and 331 intracytoplasmatic sperm injection (ICSI) pregnancies were compared with 1431 pregnancies with spontaneous conception. Alterations of serum markers were evaluated with respect to small-for-gestational-age (SGA) growth and number of embryos transferred. For pregnancy-associated plasma protein A (PAPP-A), significantly lower concentrations were observed in IVF and ICSI pregnancies compared with controls (0.86 and 0.9 versus 1.06; P<0.001). Free beta-human chorionic gonadotrophin (betaHCG) values were significantly higher in the IVF/ICSI groups than in controls (1.1 and 1.1 versus 0.94; P<0.005). IVF and ICSI pregnancies showed higher rates of SGA (10.0% and 8.2%) compared with natural conception (4.6%), but differences in PAPP-A concentrations remained significant (P<0.005) after the exclusion of SGA pregnancies. No relationship between serum values and the transfer of one, two or three embryos was observed. Centre-specific corrections may be needed to adjust screening parameters for assisted reproductive technology.

Hintergrund und Ziel: Die pineale Funktion Neugeborener ist noch nicht abschließend untersucht. Ziel dieser Studie war, bei Neugeborenen zu prüfen, ob eine neue Methode zur Speichelgewinnung verwendbar ist und ob die Bestimmung der Melatoninkonzentration (MTK) im Speichel eine zuverlässige Alternative zur Bestimmung der MTK im Serum darstellt.

Background: Because of the vast range of physiological relevant estradiol concentrations the requirements to he met by an estradiol assay are high. In the present study the performance of various commercially available estradiol assays was evaluated with regard to imprecision and long-term stability.Methods: Precision and long-term stability of 7 commercially available estradiol immunoassays were assessed in a multi-centre quality control study based on the repeated measurement of liquid BIOREF estradiol control sera by 18 laboratories during a 14-month study period.Results: The mean estradiol concentrations determined in 594 runs performed for each control level were 71 pg/ml, 349 pg/ml and 676 pg/ml. A high variation was found for the method specific mean values calculated from all results measured with the same method, which ranged between 32 - 90 pg/ml, 187 - 392 pg/ml and 373 - 790 pg/ml, resulting in a similar high inter-laboratory variation with coefficients of variation (CVs) of 25.0%, 16.7% and 17.5%. In contrast, the intra-laboratory variation of estradiol values as well as the variation of values measured with the same method were found to be considerably lower with coefficients of variation < 10% for most laboratories and methods; only the low control level was measured with CV values > 10% by the majority of laboratories and methods. For none of the laboratories a tendency was observed in the results from beginning to end of the 14 month study period indicating a high uniformity in assay production and a good long term stability of the control material used.Conclusions: The present data demonstrate that also with the currently available estradiol immunoassays the comparability of results measured with different methods is limited. With most assays very low estradiol concentrations, as observed in postmenopausal women, can be determined only with a precision which is not adequate for clinical assessment. (Clin. Lab. 2009;55:201-206)

Hintergrund: Die Physiologie von Melatonin (MT) wurde bei Neugeborenen noch nicht vollständig aufgeklärt. Das Ziel dieser Studie war, zu untersuchen, ob Neugeborene einen zirkadianen Rhythmus von MT im Serum und im Speichel haben und ob die Melatoninkonzentration (MTK) im Speichel den Tagesrhythmus von MT im Serum widerspiegelt. Methoden: 95 gesunde Neugeborene wurden nach dem Zeitpunkt der Speichelgewinnung in vier Gruppen eingeteilt (0900–1100h, 1500–1700h, 2100–2300h, 0300–0500h). Unmittelbar nach der Speichelgewinnung erfolgte die Blutentnahme im Rahmen des Neugeborenenscreenings. Ergebnisse: Bei 73 Neugeborenen konnten die Speichelproben analysiert werden. Die mediane MTK im Serum bzw. im Speichel war nicht signifikant verschieden zwischen den Gruppen: 0900–1100h: Mediane MTK 18.4pg/ml (Quartil;13.9–26.0) bzw. 10.6pg/ml (7.5–14.9), 1500–1700h: 13.3pg/ml (11.5–19.0) bzw. 9.1pg/ml (7.8–14.2), 2100–2300h: 16.0pg/ml (12.4–18.7 pg/ml) bzw. 12.3pg/ml (8.2–16.8), 0300–0500h: 13.0pg/ml (8.8–27.4) bzw. 11.2pg/ml (7.7–16.6). Die Neugeborenen zeigten weder einen zirkadianen MT-Rhythmus im Serum noch im Speichel. Die MTK im Speichel korrelierte signifikant mit der MTK im Serum in allen Gruppen (Korrelationskoeffizient nach Pearson für die Gruppen 1–4: r=0.532 [p=0.002], r=0.946 [p<0.001], r=0.788 [p<0.001], r=0.772 [p<0.001]). Schlussfolgerung: Mit dieser Studie wurde bestätigt, dass Neugeborene zum Zeitpunkt des Neugeborenenscreenings keinen MT-Rhythmus haben. Die Bestimmung der MTK im Speichel korrelierte mit der MTK im Serum zu jeder Tageszeit. Bei Neugeborenen bietet die Bestimmung von MT im Speichel eine zuverlässige, nicht invasive und schmerzfreie Alternative zur Bestimmung von MT im Serum.

Standard protocol of freezing of human ovarian tissue presupposes the very slow cooling (-0.3 C/min) from auto-seeding to -40 C, then slow cooling (-10 C/min) to -140 C and then direct plunging into liquid nitrogen. The aim of this investigation was to compare the -10 C/min cooling rate of human ovarian tissue from -40 C to -140 C with the -220 C/min cooling rate (direct plunging into liquid nitrogen) from -36 degree C. After post-thawing in vitro culture of tissue, hormonal activity as well as follicle viability was evaluated. After culture of fresh tissue pieces (Group 1), pieces after freezing and thawing with slow cooling (-10 C/min) from -40 C (Group 2) and pieces after freezing and thawing with direct plunging into liquid nitrogen (-220 C/min) from -36 C (Group 3), the supernatants showed estradiol 17-ss concentrations of 481, 441 and 459 pg per ml, respectively, and progesterone concentrations of 9.05, 5.06, 4.87 ng per ml, respectively. It is concluded that 94, 96, and 98 percent follicles for Groups 1, 2 and 3, respectively, were normal. Technique of human ovarian tissue cryopreservation with very slow cooling to -36 C and then direct plunging into liquid nitrogen with -220 C/min cooling rate is tolerated without apparent detriment.

The aim of this study was to determine the correlation between three-dimensional power Doppler sonography (3D-PDS) of the (sub)endometrium and concentrations of angiogenic cytokines in patients attending an IVF programme. A total of 42 patients was included in a prospective, non-randomized clinical study. 3D-PDS of the (sub)endometrium was performed on the day of oocyte aspiration, with and without contrast agent. Quantitative assessment included the following 3D Doppler parameters: vascularization index, flow intensity, and vascularization flow index. On the same day, concentrations of oestradiol (serum only), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF) 1, IGF-binding protein 3 (IGFBP-3) and leptin were determined in the serum and in the follicular fluid. All 3D-PDS indices were significantly higher with contrast enhancement (P < 0.05). Follicular fluid concentrations of VEGF and IGFBP-3, as well as serum concentrations of leptin, showed significant P-values when correlated with (sub)endometrial Doppler indices. A weak linear dependency appeared between flow intensity and VEGF and leptin. Furthermore, weak dependencies were apparent between 3D Doppler parameters and high follicular fluid concentrations of VEGF and IGFBP-3. It is concluded that there is only little evidence for an association between (sub)endometrial Doppler indices as assessed by 3D-PDS and concentrations of angiogenic cytokines.