Arieh Cohen

The prevalence of polycystic ovary syndrome in a normal population according to the Rotterdam criteria versus revised criteria including anti-Müllerian hormone

STUDY QUESTION What is the prevalence in a normal population of polycystic ovary syndrome (PCOS) according to the Rotterdam criteria versus revised criteria including anti-Müllerian hormone (AMH)? SUMMARY ANSWER The prevalence of PCOS was 16.6% according to the Rotterdam criteria. When replacing the criterion for polycystic ovaries by antral follicle count (AFC) > 19 or AMH > 35 pmol/l, the prevalence of PCOS was 6.3 and 8.5%, respectively. WHAT IS KNOWN ALREADY?: The Rotterdam criteria state that two out of the following three criteria should be present in the diagnosis of PCOS: oligo-anovulation, clinical and/or biochemical hyperandrogenism and polycystic ovaries (AFC ≥ 12 and/or ovarian volume >10 ml). However, with the advances in sonography, the relevance of the AFC threshold in the definition of polycystic ovaries has been challenged, and AMH has been proposed as a marker of polycystic ovaries in PCOS. STUDY DESIGN, SIZE, DURATION From 2008 to 2010, a prospective, cross-sectional study was performed including 863 women aged 20-40 years and employed at Copenhagen University Hospital, Rigshospitalet, Denmark. PARTICIPANTS/MATERIAL, SETTING, METHODS We studied a subgroup of 447 women with a mean (±SD) age of 33.5 (±4.0) years who were all non-users of hormonal contraception. Data on menstrual cycle disorder and the presence of hirsutism were obtained. On cycle Days 2-5, or on a random day in the case of oligo- or amenorrhoea, sonographic and endocrine parameters were measured. MAIN RESULTS AND THE ROLE OF CHANCE The prevalence of PCOS was 16.6% according to the Rotterdam criteria. PCOS prevalence significantly decreased with age from 33.3% in women < 30 years to 14.7% in women aged 30-34 years, and 10.2% in women ≥ 35 years (P < 0.001). In total, 53.5% fulfilled the criterion for polycystic ovaries with a significant age-related decrease from 69.0% in women < 30 years to 55.8% in women aged 30-34 years, and 42.8% in women ≥ 35 years (P < 0.001). AMH or age-adjusted AMH Z-score was found to be a reliable marker of polycystic ovaries in women with PCOS according to the Rotterdam criteria [area under the curve (AUC) 0.994; 95% confidence interval (CI): 0.990-0.999] and AUC 0.992 (95% CI: 0.987-0.998), respectively], and an AMH cut-off value of 18 pmol/l and AMH Z-score of -0.2 showed the best compromise between sensitivity (91.8 and 90.4%, respectively) and specificity (98.1 and 97.9%, respectively). In total, AFC > 19 or AMH > 35 occurred in 17.7 and 23.0%, respectively. The occurrence of AFC > 19 or AMH > 35 in the age groups < 30, 30-34 and ≥ 35 years was 31.0 and 35.7%, 18.8 and 21.3%, and 9.6 and 18.7%, respectively. When replacing the Rotterdam criterion for polycystic ovaries by AFC > 19 or AMH > 35 pmol/l, the prevalence of PCOS was 6.3 or 8.5%, respectively, and in the age groups < 30, 30-34 and ≥ 35 years, the prevalences were 17.9 and 22.6%, 3.6 and 5.6%, and 3.6 and 4.8%, respectively. LIMITATIONS, REASON FOR CAUTION The participants of the study were all health-care workers, which may be a source of selection bias. Furthermore, the exclusion of hormonal contraceptive users from the study population may have biased the results, potentially excluding women with symptoms of PCOS. WIDER IMPLICATIONS OF THE FINDINGS AMH may be used as a marker of polycystic ovaries in PCOS. However, future studies are needed to validate AMH threshold levels, and AMH Z-score may be appropriate to adjust for the age-related decline in the AFC. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER Not applicable.

Phthalates and Perfluorooctanesulfonic Acid in Human Amniotic Fluid: Temporal Trends and Timing of Amniocentesis in Pregnancy

Background: Measures of prenatal environmental exposures are important, and amniotic fluid levels may directly reflect fetal exposures during hypothesized windows of vulnerability. Objectives: We aimed to detect various phthalate metabolites and perfluorooctanesulfonic acid (PFOS) in human amniotic fluid, to study temporal exposure trends, and to estimate potential associations with gestational week of amniocentesis and maternal age and parity at amniocentesis. Methods: We studied 300 randomly selected second-trimester amniotic fluid samples from a Danish pregnancy-screening biobank covering 1980 through 1996. We used only samples from male offspring pregnancies. We assayed the environmental pollutants by liquid chromatography/triple quadrupole mass spectrometry and analyzed data using generalized linear regression models. Results: We detected the di(2-ethylhexyl) phthalate (DEHP) metabolite mono(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP) at a median concentration of 0.27 ng/mL [interquartile range (IQR): 0.20–0.37 ng/mL], the diisononyl phthalate (DiNP) metabolite mono(4-methyl-7-carboxyheptyl) phthalate (7cx-MMeHP) at 0.07 ng/mL (IQR: 0.05–0.11 ng/mL), and PFOS at 1.1 ng/mL (IQR: 0.66–1.60 ng/mL). An increase of 1 calendar year was associated with 3.5% lower [95% confidence interval (CI): –4.8%, –2.1%] 5cx-MEPP levels and with 7.1% higher (95% CI: 5.3%, 9.0%) 7cx-MMeHP levels. For each later gestational week of amniocentesis, 5cx-MEPP was 9.9% higher (95% CI: 4.8%, 15.2%), 7cx-MMeHP was 8.6% higher (95: CI: 2.7%, 14.9%), and PFOS was 9.4% higher (95: CI: 3.3%, 15.9%). We observed no associations with maternal age or parity. Conclusions: Measured metabolite levels appeared to parallel decreasing DEHP exposure and increasing DiNP exposure during the study period. The environmental pollutant levels were positively associated with later gestational age at amniocentesis during pregnancy weeks 12–22.

RE: Is There a Correlation Between Androgens and Sexual Desire in Women?

We were very interested to read the results of the study by WåhlinJacobsen et al. [1] titled “Is there a correlation between androgens and sexual desire in women?” The researchers used mass spectrometry methods to investigate age-adjusted serum levels of dehydroepiandrosterone sulfate (DHEA-S), androstenedione, and testosterone, as well as androsterone glucuronide (ADT-G)—a measure of total androgen activity. The results were consistent with our published study of 121 women with and 124 women without a diagnosis of Hypoactive Sexual Desire Disorder (using the definition of the Diagnostic and Statistical Manual, 4th edition, text-revised), where no group differences in androgen metabolites were found [2]. We were somewhat perplexed by the apparent change in Wåhlin-Jacobsen et al.’s [1] objective in their paper from investigating a possible correlation between serum levels of testosterone and/or androgen metabolites and sexual desire, to finding a measure that does indeed correlate with desire and to apparently suggest its use hereon. The conclusion in the abstract that ADT-G did not correlate more strongly than circulating androgens with sexual desire and therefore is not superior to measuring circulating androgens with mass spectrometry presupposes an established correlation between androgens and sexual desire. The authors note that “measuring the degradation product of the intracellular testosterone turnover should therefore yield a better estimate of both the intracellular turnover and the total activity of androgens. In contrast to what we expected, however, no statistically significant correlations were established between ADT-G and sexual desire. Therefore, based on these current and previous results, we cannot conclude that ADT-G is a better biomarker of the link between androgens and women’s sexual desire than measuring the amount of circulating androgens.” (p. 12). An explanation for these findings can, however, be based upon the understanding that the metabolite of androgens chosen as a parameter of total androgenic activity, namely ADT-G, although a logical suggestion, could have to take into account a more complex pattern than originally believed [3]. As mentioned by the authors, it could well be that ADT-G represents only a fraction of all androgen metabolites. In fact, it could be that ADT-sulfate and epi-ADT-G (and likely others) could represent a much larger proportion of androgen metabolites than ADT-G alone. However, since the very low serum levels of testosterone observed in women during their whole life essentially result from some leakage of the intracellular testosterone synthesized locally from DHEA [4], it might be reasonable, at the present time, to measure the precursors of intracellular testosterone, namely serum DHEA, DHEA-S, androstene-3β,17β-diol, and androstenedione. In fact, low serum levels of all these four androgen precursors have been correlated with sexual dysfunction [1,2,5]. The authors did find a correlation between total testosterone, free testosterone, androstenedione, and DHEA-S in the subgroup of women aged 25–44 who were not using any hormonal contraception (n = 168). In our study, 246 women were more stringently recruited to exclude confounds such as any medication potentially altering sexual response and desire, scoring in the clinically significant depressed range on a validated measure of depression, significant relationship conflict, and sexual pain, and this study failed to show a correlation between mass spectrometry-measured serum testosterone and desire but showed a correlation with low serum DHEA-S [2]. We maintain our conclusion [2] that in large samples of women, there is not yet conclusive evidence of a significant correlation between accurately measured total testosterone activity and sexual desire. This is not to say androgen activity may not be important, but at this point, such evidence of a correlation has not been empirically supported. As indicated above, it could well be that serum testosterone is not a reliable marker since it represents only leakage of intracellular testosterone made by the intracellular mechanisms of intracrinology [4]. The conclusion of Wåhlin-Jacobsen et al. [1] that as measurement of ADT-G showed no significant correlation with women’s sexual desire, such measurement “did not seem to be an improvement over measuring circulating androgens with MS” (p. 14) suggests that there was a very different a priori objective to the study than what was published. A recent editorial reminds us of the significant dangers inherent to modification of a priori hypotheses and the threat to statistical analyses as well as interpretation of the findings [6]. That the identification/measurement of the most appropriate metabolites of androgens has not yet been determined in no way negates the importance of intracellular testosterone production. We offer this analogy: the hypothetical investigation of a correlation between intake of vitamin C supplements and susceptibility to influenza. Imagine researchers counting vitamin C supplements taken by patients and finding the numbers to correlate negatively with number of influenza infections. However, when measuring their serum vitamin C levels, no correlation is found between vitamin C levels and cases of influenza. If the researchers then said “from here-on just count vitamin C supplements as this correlates better with influenza susceptibility,” perhaps the lack of a scientific demonstration would be more obvious. In summary, with the strong caveat of relatively lax exclusion criteria, the findings by Wåhlin-Jacobsen et al. have added to the literature on women’s sexual desire and androgens, which are based on the currently available most optimal assays for serum androgens and precursor hormones (liquid chromatography-mass spectrometry), and for total (intracellular plus serum) androgens (ADT-G). As in our study [2], precursors of intracellular testosterone correlated with sexual desire in women aged 25–65 years, none of whom were taking systemic sex hormones. In contrast to our study, in the subgroup of 168 women aged 25–45 years, desire also correlated with measures of serum testosterone. It will be important to repeat these measures on this targeted subgroup, but with stricter exclusion criteria as well as the entry criterion of a diagnosis of sexual interest/arousal disorder assessed by detailed interview as currently available questionnaires do not reflect current conceptualization of women’s sexual response or current 1

Routine analysis of alcohol and nonylphenol polyethoxylates in wastewater and sludge using liquid chromatography-electrospray mass spectrometry.

A wide range of alcohol and nonylphenol polyethoxylates was determined by separation on a reversed-phase liquid chromatographic column, followed by electrospray ionisation-mass spectral analysis. The compounds were separated chromatographically according to their aliphatic chain length. The mass spectral analysis functioned as a second separation step during which homologues of the ethoxylates were separated according to their polyethoxylate chain length. In this manner a truly orthogonal separation was obtained. The compounds were detected as ammonium complexes. The analysis presented is capable of qualitative and quantitative determination of a large number of ethoxylates as well as their metabolites in a single run. The method was applied to many different sample types, ranging from primary and treated wastewater to sludge. Batches of 50 real samples were routinely analysed without the need for cleaning the mass spectrometer or regeneration of the column. By utilising the extracted mass chromatograms, detection limits of 1 to 10 microg/l could be obtained for individual compounds in water samples, while the detection limits were around 100 microg/kg in sludge, depending on the degree of pollution.

High-performance anion-exchange chromatography-electrospray mass spectrometry for investigation of the substituent distribution in hydroxypropylated potato amylopectin starch.

The use of high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) coupled on-line with electrospray mass spectrometry (ESI-MS) for analysis of the substitution pattern in chemically modified starch, has been investigated. In order to characterise the distribution of substitution groups along the polymer chain, hydroxypropylated potato amylopectin starch (HPPAP) was subjected to enzymic hydrolysis, followed by analysis of the degradation products by HPAEC-PAD-MS. When using conventional chromatographic techniques for characterisation of enzymic hydrolysates, standard compounds are required for identification of the hydrolysis products. However, the on-line coupling with ESI-MS allowed identification of all products obtained, substituted as well as unsubstituted, and also of those compounds that co-eluted, without the need for standards. Further, HPAEC-PAD-MS was shown to be useful for analysis of the substitution pattern in modified starch; from results obtained it was suggested that the hydroxypropyl groups were homogeneously distributed in the amylopectin molecule. It was also shown that the starch hydrolysing enzymes were hindered by the hydroxypropyl groups and preferentially cleaved glucosidic linkages between unsubstituted glucose units.

Vitamin D Status during Pregnancy and the Risk of Subsequent Postpartum Depression: A Case-Control Study

Epidemiological studies have provided evidence of an association between vitamin D insufficiency and depression and other mood disorders, and a role for vitamin D in various brain functions has been suggested. We hypothesized that low vitamin D status during pregnancy might increase the risk of postpartum depression (PPD). The objective of the study was thus to determine whether low vitamin D status during pregnancy was associated with postpartum depression. In a case-control study nested in the Danish National Birth Cohort, we measured late pregnancy serum concentrations of 25[OH]D3 in 605 women with PPD and 875 controls. Odds ratios [OR) for PPD were calculated for six levels of 25[OH]D3. Overall, we found no association between vitamin D concentrations and risk of PPD (p = 0.08). Compared with women with vitamin D concentrations between 50 and 79 nmol/L, the adjusted odds ratios for PPD were 1.35 (95% CI: 0.64; 2.85), 0.83 (CI: 0.50; 1.39) and 1.13 (CI: 0.84; 1.51) among women with vitamin D concentrations < 15 nmol/L, 15–24 nmol/L and 25–49 nmol/L, respectively, and 1.53 (CI: 1.04; 2.26) and 1.89 (CI: 1.06; 3.37) among women with vitamin D concentrations of 80–99 nmol/L and ≥ 100 nmol/L, respectively. In an additional analysis among women with sufficient vitamin D (≥ 50 nmol/L), we observed a significant positive association between vitamin D concentrations and PPD. Our results did not support an association between low maternal vitamin D concentrations during pregnancy and risk of PPD. Instead, an increased risk of PPD was found among women with the highest vitamin D concentrations.

Vitamin D Measured in Maternal Serum and Offspring Neurodevelopmental Outcomes: A Prospective Study with Long-Term Follow-Up

Background: Vitamin D is obtained from dietary sources and synthesized in the skin during exposure to ultraviolet B radiation in sunlight. During pregnancy, vitamin D is transported from mother to fetus through the placenta in the form of 25-hydroxyvitamin D [25(OH)D]. There is evidence that vitamin D influences neuronal differentiation, endocrine functions, and fetal brain growth. Animal studies indicate alterations in the offspring brain as a consequence of vitamin D deficiency during pregnancy. In humans, maternal vitamin D insufficiency has been linked to impaired child language development. Using data from a prebirth cohort with up to 22 years of follow-up, we examined the association of vitamin D status with proxies of offspring neurodevelopmental outcomes. During 1988-1989, pregnant women were recruited for the DaFO88 cohort (n = 965) in Aarhus, Denmark. Maternal concentrations of 25(OH)D were quantified in serum from week 30 of gestation via the LC-MS/MS method (n = 850). Offspring were followed up through national registries until the age of 22 years. We evaluated the association of the maternal concentration of 25(OH)D with offspring neurodevelopmental outcomes defined as first admission diagnosis or prescription of medication for (1) ADHD, (2) depression, and (3) scholastic achievement based on the mean grade on standardized written examinations in the 9th grade (final exams after 10 years of compulsory school in Denmark). Key Messages: Maternal concentrations of 25(OH)D were higher compared to current levels (median 76 nmol/l; 5th to 95th percentiles 23-152). There was a direct association between maternal vitamin D status and offspring depression (ptrend = 0.01); for ADHD there was no association. Scholastic achievement was slightly higher for offspring of mothers with a vitamin D status in the range of >50-125 nmol/l, but this nonlinear association was not statistically significant. Conclusions: Our analyses based on biomarker measurement of 25(OH)D from a cohort of 850 pregnant women combined with long-term follow-up showed no support for a beneficial fetal programming effect of vitamin D status with regard to behavioral and affective disorders and scholastic achievement.

Biochemical screening of 504,049 newborns in Denmark, the Faroe Islands and Greenland — Experience and development of a routine program for expanded newborn screening

Expanded newborn screening for selected inborn errors of metabolism (IEM) in Denmark, the Faroe Islands and Greenland was introduced in 2002. We now present clinical, biochemical, and statistical results of expanded screening (excluding PKU) of 504,049 newborns during nine years as well as diagnoses and clinical findings in 82,930 unscreened newborns born in the same period. The frequencies of diagnoses made within the panel of disorders screened for are compared with the frequencies of the disorders in the decade preceding expanded newborn screening. The expanded screening was performed as a pilot study during the first seven years, and the experience obtained during these years was used in the development of the routine neonatal screening program introduced in 2009. Methods for screening included tandem mass spectrometry and an assay for determination of biotinidase activity. A total of 310 samples from 504,049 newborns gave positive screening results. Of the 310 results, 114 were true positive, including results from 12 newborns in which the disease in question was subsequently diagnosed in their mothers. Thus, the overall frequency of an IEM in the screening panel was 1:4942 (mothers excluded) or 1:4421 (mothers included). The false positive rate was 0.038% and positive predictive value 37%. Overall specificity was 99.99%. All patients with true positive results were followed in The Center for Inherited Metabolic Disorders in Copenhagen, and the mean follow-up period was 45 months (range 2109 months). There were no deaths among the 102 children, and 94% had no clinically significant sequelae at last follow-up. Our study confirms the higher frequency of selected IEM after implementation of expanded newborn screening and suggests an improved outcome for several disorders. We argue that newborn screening for these disorders should be standard of care, though unresolved issues remain, e.g. about newborns with a potential for remaining asymptomatic throughout life. Well organized logistics of the screening program from screening laboratory to centralized, clinical management is important.

Development and Validation of a Vitamin D Status Prediction Model in Danish Pregnant Women: A Study of the Danish National Birth Cohort

Vitamin D has been hypothesized to reduce risk of pregnancy complications such as preeclampsia, gestational diabetes mellitus, and preterm delivery. However, many of these outcomes are rare and require a large sample size to study, representing a challenge for cohorts with a limited number of preserved samples. The aims of this study were to (1) identify predictors of serum 25-hydroxy-vitamin D (25(OH)D) among pregnant women in a subsample (N = 1494) of the Danish National Birth Cohort (DNBC) and (2) develop and validate a score predicting 25(OH)D-status in order to explore associations between vitamin D and maternal and offspring health outcomes in the DNBC. In our study sample, 42.3% of the population had deficient levels of vitamin D (<50 nmol/L 25(OH)D) and average levels of 25(OH)D-status were 56.7(s.d. 24.6) nmol/L. A prediction model consisting of intake of vitamin D from diet and supplements, outdoor physical activity, tanning bed use, smoking, and month of blood draw explained 40.1% of the variance in 25(OH)D and mean measured 25(OH)D-level increased linearly by decile of predicted 25(OH)D-score. In total 32.2% of the women were placed in the same quintile by both measured and predicted 25(OH)D-values and 69.9% were placed in the same or adjacent quintile by both methods. Cohens weighted kappa coefficient (Κ = 0.3) reflected fair agreement between measured 25(OH)D-levels and predicted 25(OH)D-score. These results are comparable to other settings in which vitamin D scores have shown similar associations with disease outcomes as measured 25(OH)D-levels. Our findings suggest that predicted 25(OH)D-scores may be a useful alternative to measured 25(OH)D for examining associations between vitamin D and disease outcomes in the DNBC cohort, but cannot substitute for measured 25(OH)D-levels for estimates of prevalence.

Neonatal vitamin D status and risk of multiple sclerosis A population-based case-control study

Objective: As previous research has suggested that exposure to vitamin D insufficiency in utero may have relevance for the risk of multiple sclerosis (MS), we aimed to examine the direct association between level of neonatal vitamin D and risk of MS. Methods: We carried out a matched case-control study. Dried blood spots samples (DBSS) belonging to 521 patients with MS were identified in the Danish Newborn Screening Biobank. For every patient with MS, 1–2 controls with the same sex and birth date were retrieved from the Biobank (n = 972). Level of 25-hydroxyvitamin D (25[OH]D) in the DBSS was measured using liquid chromatography tandem mass spectroscopy. The association between different levels of 25(OH)D and risk of MS was evaluated by odds ratios (OR) calculated in conditional logistic regression models. Results: We observed that lower levels of 25(OH)D in neonates were associated with an increased risk of MS. In the analysis by quintiles, MS risk was highest among individuals in the bottom quintile (<20.7 nmol/L) and lowest among those in the top quintile of 25(OH)D (≥48.9 nmol/L), with an OR for top vs bottom of 0.53 (95% confidence interval [CI] 0.36–0.78). In the analysis treating 25(OH)D as a continuous variable, a 25 nmol/L increase in neonatal 25(OH)D resulted in a 30% reduced risk of MS (OR 0.70, 95% CI 0.57–0.84). Conclusion: Low concentrations of neonatal vitamin D are associated with an increased risk of MS. In light of the high prevalence of vitamin D insufficiency among pregnant women, our observation may have importance for public health.