Victor H.H. Goh

Reexamination of Testosterone, Dihydrotestosterone, Estradiol and Estrone Levels across the Menstrual Cycle and in Postmenopausal Women Measured by Liquid Chromatography Tandem Mass Spectrometry

Measuring serum androgen levels in women has been challenging due to limitations in method accuracy, precision sensitivity and specificity at low hormone levels. The clinical significance of changes in sex steroids across the menstrual cycle and lifespan has remained controversial, in part due to these limitations. We used validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays to determine testosterone (T) and dihydrotestosterone (DHT) along with estradiol (E2) and estrone (E1) levels across the menstrual cycle of 31 healthy premenopausal females and in 19 postmenopausal females. Samples were obtained in ovulatory women in the early follicular phase (EFP), midcycle and mid luteal phase (MLP). Overall, the levels of T, DHT, E2 and E1 in premenopausal women measured by LC-MS/MS were lower overall than previously reported with immunoassays. In premenopausal women, serum T, free T, E2, E1 and SHBG levels peaked at midcycle and remained higher in the MLP, whereas DHT did not change. In postmenopausal women, T, free T, SHBG and DHT were significantly lower than in premenopausal women, concomitant with declines in E2 and E1. These data support the hypothesis that the changes in T and DHT that occur across the cycle may reflect changes in SHBG and estrogen, whereas in menopause, androgen levels decrease. LC-MS/MS may provide more accurate and precise measurement of sex steroid hormones than prior immunoassay methods and can be useful to assess the clinical significance of changes in T, DHT, E2 and E1 levels in females.

Simultaneous Measurement of Serum Testosterone and Dihydrotestosterone by Liquid Chromatography–Tandem Mass Spectrometry

BACKGROUND Recent reports have described inherent problems with androgen immunoassays compared with mass spectrometry analyses. METHODS We developed a method for measuring serum testosterone (T) and 5alpha-dihydrotestosterone (DHT) simultaneously via liquid-liquid extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with positive-mode electrospray ionization. RESULTS The DHT and T calibrators showed a linear response from 0.069 nmol/L to 34.4 nmol/L and 69.3 nmol/L, respectively. T interference in the DHT assay and vice versa were negligible. Within- and between-run imprecision values were <5% for both analytes. Percent recoveries of T and DHT spiked into samples at concentrations spanning the calibration curve were 100%-113% and 98%-107%, respectively. The lower limit of quantification was 0.069 nmol/L for both steroids. Serum T concentrations measured by LC-MS/MS were different from those obtained by RIA, especially at lower T concentrations. Serum DHT concentrations measured by LC-MS/MS were markedly lower than those generated by RIA because of the nonselectivity of the RIA without chromatography. The reference intervals (mean +/- 2 SDs) determined for T and DHT were 9.2-33.7 nmol/L and 0.47-2.65 nmol/L, respectively, for 113 healthy adult men and 0.33-2.02 nmol/L and 0.09-0.91 nmol/L, respectively, for 133 healthy premenopausal women. CONCLUSIONS We have developed and validated a selective and precise method for simultaneous measurements of serum T and DHT that can be adopted for routine measurements of these androgens in health and disease in men and women.

Validation of a Testosterone and Dihydrotestosterone Liquid Chromatography Tandem Mass Spectrometry Assay: Interference and Comparison with Established Methods

Testosterone (T) and its metabolite dihydrotestosterone (DHT) are androgens with different biologic profiles. T and DHT measurements are required for assessment of patients with ambiguous genitalia, hirsutism, during 5 alpha reductase treatment of prostate disorders, and new androgen formulations. Our laboratory has developed and validated a method to simultaneously measure serum T and DHT with liquid chromatography tandem mass spectrometry (LC-MS/MS) for use in a clinical chemistry laboratory. Analysis of sera from blood collected in tubes containing clot activator gave results of T that were fourfold higher than blood collected in plain tubes. Changing the ion pair selected for monitoring eliminated this interference by clot activators. Blood collected in fluoride-coated tubes gave serum T and DHT levels that were 20 and 15% lower, respectively than levels measured in blood collected in plain tubes (no additives). Addition of T enanthate to blood collected in plain tubes caused a dose related increase serum T levels due to the action of non-specific esterases in the red cells. This esterase activity could be avoided by using fluoride tubes for blood collection. Serum DHT levels were consistently lower when measured by LC-MS/MS versus radioimmunoassay. The differences were concentration dependent and the variance for the difference was large when serum DHT concentration was low. Celite chromatograph prior to radioimmunoassay reduced the differences between the two methods, thus confirming that higher levels of DHT obtained by immunoassays were probably due to interfering substances which were partially removed by Celite chromatography.

Sleep, Sex Steroid Hormones, Sexual Activities, and Aging in Asian Men

This was a cross-sectional study to examine the different associations of age and sleep duration with sex steroid hormones and sexual activities in 531 Asian Chinese men aged between 29 and 72 years old. Sleep duration and sexual activities were evaluated through a self-administered questionnaire, and total testosterone (T), sex hormone-binding globulin (SHBG), estradiol (E2), and dehydroepiandrosterone sulfate (DHEAS) were measured by established immunoassay methods in a single blood sample collected between 8:00 and 11:00 am. Bioavailable T (BioT) was calculated using the Vermeulen formula. Age was a major determinant of sleep, sex steroid hormones, and sexual activities in men. BioT, DHEAS, coital frequency, masturbation, and sleep duration declined with age. On the other hand, SHBG and E2 increased with age. Sleep duration, independently of age, aerobic exercise, and body fat, was positively associated with T and BioT, but not with DHEAS, E2, or any of the sexual activities studied. Men who masturbated had higher levels of both T and BioT. DHEAS was significantly associated with coital frequency and desire for sex. The present study showed that besides age, sleep duration was associated with androgen concentrations in men, and thus the evaluation of sleep hygiene may be beneficial in the management of men with low androgen concentrations. DHEAS may be independently associated with some sexual functions in men.

Sex and aging in the city: Singapore

A survey was carried out on 307 healthy men and 774 women, aged between 30 and 70 years to evaluate the determinants of sex life in the highly urbanized Singapore population. The results showed that significantly more women (28.8%) as compared to men (16.3%) were currently sexually inactive. The main reason for sexual inactivity for women was being divorced or widowed (37.9%) and for men, the loss of interest in sex (42.8%). Relationship problems and being stressed out in life were the next two most common reasons for both men and women. Coital frequency for men and women between the ages of 30 and 55 years averaged about five to six times monthly and was significantly reduced to about three times monthly in those above 55 years old. More men (25.7%) wanted to have more frequent sex than women (5.4%), but were unable to fulfill their desire and the primary reason was that they were too stressed out in life. Results from this study revealed that sex life is determined not only by physiological, but also cultural, social and lifestyle factors. In Singapore, lifestyle factors accounted largely for sexual inactivity, and the inability to fulfill the desire for more frequent sex. The term ‘lifestyle impotency’ was coined to describe the group of men and women who were too stressed out in life to have sex. Therefore, there is a need to evolve a paradigm for the management of sexual dysfunctions by taking into account the underlying etiological factors which, as we have shown, may not be resolved by pharmaceutical interventions.

Effects of hyperstimulation with gonadotrophins and age of females on oocytes and their metaphase II status in rats

The present study was undertaken to evaluate the effects of hyperstimulation and aging on the number and proportion of oocytes in the metaphase II stage in female Wistar rats. It explored the validity of the hypothesis that a combination of hyperstimulation with pregnant mare serum gonadotrophins (PMSG) and age could compromise, to a greater extent, the oocyte quality as indicated by the proportion of ovulated oocytes in the metaphase II stage. Female Wistar rats were stimulated with varying doses of PMSG and human chorionic gonadotrophins (hCG) and the number and proportion of ovulated oocytes in the metaphase II stage were examined and compared between different groups of young adult (8–10 weeks old) and aging (30–32 weeks old) female rats. While spontaneous ovulation occurred in all young adult rats, only 50% of the aging rats did. The ovulation rate in aging rats was increased from 50 to 93% when non‐PMSG‐stimulated rats were given a dose of 10 IU of hCG at proestrus. The lower number of ovulated oocytes noted, even in those hyperstimulated with high doses of PMSG/hCG, also indicated a reduction in fertility in aging rats. Under the influence of high doses of PMSG, all aging rats ovulated, but as with the young adult rats, a higher dose of hCG was needed to achieve the maximum number of ovulated oocytes from the PMSG‐induced expanded pool of preovulatory follicles. However, the average number of ovulated oocytes in aging rats was, nevertheless, still significantly lower than in young adult rats even when approximation of weight was considered. No consistent significant difference in proportion of normal oocytes was noted within groups and between young adult and aging rats. A lower proportion of ovulated oocytes was arrested at the metaphase II stages when rats, whether they were young adult or aging, were hyperstimulated with 40 IU of PMSG. However, this proportion was restored to normal (about 100%) when a higher dose of hCG, which is a signal responsible for initiating oocyte maturation, was used. Results of the present study showed that there appears to be an age‐related reduction of sensitivity of the preovulatory follicles to the ovulation induction signal of hCG and thus higher doses of hCG were needed to ovulate the PMSG‐induced expanded pool of dominant follicles. In older rats, apart from the obvious depletion of the pool of follicles, the evidence from the present study suggests that some of these older rats do have follicles, but that these were unable to develop to preovulatory follicles, probably because of the absence of sufficiently high levels of gonadotrophins essential for the initiation of folliculogenesis. PMSG‐hyperstimulation can affect nuclear maturation; the proportion of ovulated oocytes not arrested at the metaphase II stage was higher. However, the proportion of ovulated oocytes at the metaphase II was restored to normal by increasing the dose of hCG use. Hence, meiotic aberration in rats is not age‐dependent but rather dependent on the amplitude of the luteinizing hormone (LH)/hCG surge present. The results from this study nullified the hypothesis that hyperstimulation in combination with aging would lead to a higher proportion of abnormality in ovulated oocytes with respect to their being at inappropriate meiotic stages. Mol. Reprod. Dev. 55:104–108, 2000.

Aging in Asia: A cultural, socio-economical and historical perspective

Asia has about 60% of the world population and population aging is occurring more rapidly in Asia than in Western countries. The group aged 65 years and above will increase from 207 million in 2000 to 857 million in 2050, a staggering increase of 314%. The diversity in economic, demographic, religious, cultural and geo-political factors in Asia is unparalleled by any other continent, and is, in part, contributory to the rapid rise in population aging. By 2050, those under 15 years old will have shrunk from 30% in 2000 to 19%, while those aged 65 years and above will increase from 6% to 18%. In addition, the gender divide still persists with 100 elderly women to 70 elderly men. These projected demographic changes pose three major challenges: 1) how best to address the rising population of the group aged 65 years and above, 2) how to address the shrinking population of the young as well as the working adults, and 3) how to address the problems arising from the disproportionate increase in older women than men. From now to 2050, it will be expeditious for each country in Asia to look into ways of reversing the decline in total fertility rates (TFRs) and restore to replacement levels. If not, at least introduce measures to halt its free fall. Due to the complexity of factors that have influenced the fall in TFRs in Asia, it will be a daunting task to reverse this fall. There is no “single size fits all” solution to this complex problem. Research work in this short-term strategy in addressing the aging population is urgent. In the longer term, the East-West Centre have suggested four modalities, 1) establish policies and programmes that enhance traditional Asian systems of family support for the elderly; 2) introduce policy reform that encourages the elderly who are still capable of remaining in the work force; 3) create institutions and systems that support high levels of personal saving; and 4) formulate public programmes, including pension schemes and national healthcare systems. We need to work while there is time.

Association between an intronic apolipoprotein E polymorphism and bone mineral density in Singaporean Chinese females.

INTRODUCTION Apolipoprotein E (ApoE) is implicated in the pathogenesis of osteoporosis. OBJECTIVE To investigate possible association of the non-classical APOE gene +113C/G (rs440446) intron 1 enhancer polymorphism with bone mineral density (BMD) in a homogeneous Chinese population in Singapore. METHODS A total of 655 volunteers, males and females, aged between 31 and 72 years, from the public participated. BMD was measured using dual-energy X-ray absorptiometry and APOE +113C/G (rs440446) genotypes were determined by Sequenom MassARRAY system. To adjust for potential confounders, anthropometric, demographic, and lifestyle determinants were obtained, and serum lipids and E(2) were measured. RESULTS The +113C/G (rs440446) polymorphism within the APOE gene was associated with BMD in Chinese Singaporean females only. Females with the heterozygous CG genotype were significantly associated with reduced total, lumbar spine, and femoral neck of hip BMD, after multilevel adjustment of confounders. The association was stronger in the spine than in the hip. When females were stratified according to WHO classification for osteoporosis, those with CG and GG genotypes had increased risk (OR 3.50 and 2.22, respectively) of developing osteopenia/osteoporosis in the lumbar spine. Serum lipids did not explain the influence of APOE +113 C/G (rs440446) on BMD. CONCLUSION This study demonstrated an association between APOE +113C/G (rs440446) polymorphism with measures of BMD in Singaporean Chinese females.

The moderating impact of lifestyle factors on sex steroids, sexual activities and aging in Asian men

The present study sought to evaluate the relative associations of exercise, sleep and other lifestyle habits with aging, sex hormones, percent body fat (%BF) and sexual activities in men living in the community. A better understanding of this complex interrelationship is important in helping the formulation of modalities for a holistic approach to the management of aging men. The results showed that age is a major determinant for many physiological parameters, including sleep, hormonal and metabolic parameters, some lifestyle factors and sexual activities. Testosterone (T), bioavailable testosterone (BioT) and dehydroepiandrosterone sulphate (DHEAS) concentrations decreased with age, while estradiol (E2), sex hormone-binding globulin (SHBG) and %BF increased with age. In addition, there exist intricate associations among hormonal and lifestyle factors, %BF and age. High-intensity exercise and longer duration of sleep were associated with higher concentrations of T and BioT. T was shown to be associated positively with men who were engaged in masturbation. DHEAS was associated with men wanting more sex and with good morning penile rigidity. Older Singaporean men tended to sleep for shorter duration, but exercised more intensely than younger men. Coital and masturbation frequencies decreased with age, and a significantly greater number of younger men were engaged in masturbation. Relationship between the partners is a key determinant of sexuality in men. It appears that T may have a limited, while dehydroepiandrosterone (DHEA) have a greater role than previously suggest, as a motivational signal for sexual function in men. Both biological and psychosocial factors interact with each other to influence sexual functions in men. Hence, a biopsychosocial approach may be more appropriate for a more lasting resolution to sexual dysfunctions in men.

Changes in body composition, and endocrine and metabolic functions in healthy elderly Chinese men following growth hormone therapy

Background An age-related decline in growth hormone (GH) level has been established, and this decline is associated with changes in body composition as well as a general increase in susceptibility to illness and a reduced sense of well-being. The current study, a first in Asia, sought to examine the effects of GH therapy on body composition and other endocrine and metabolic functions in a group of healthy elderly Chinese men.Methods A total of 23 healthy elderly Chinese men, aged between 60 and 69 years, were injected subcutaneously, three times weekly, with 0.08 U/kg of recombinant GH for 6 months. Various hormones and biochemical parameters, together with percentage lean body mass and body fat, were measured before, 3 and 6 months after the start and 3 months after the cessation of GH therapy.Results A significant increase in lean body mass, up to 9.1% over baseline values at 3 months post-therapy, and a significant decrease in body fat, up to 3.1%, were noted. GH therapy also induced variable and signi...