Can anti-Müllerian hormone concentrations be used to determine gonadotrophin dose and treatment protocol for ovarian stimulation?

The ability to predict the response potential of women to ovarian stimulation may allow the development of individualized ovarian stimulation protocols. This tailored approach to ovarian stimulation could reduce the incidence of ovarian hyperstimulation syndrome in women predicted to have an excessive response to stimulation or could improve pregnancy outcomes in women classed as poor responders. Namely, variation of the type of gonadotrophin-releasing hormone (GnRH) analogue or the form and dosage of gonadotrophin used for stimulation could be adjusted according to an individual's response potential. The serum concentration of anti-Müllerian hormone (AMH) is established as a reliable marker of ovarian reserve, with decreasing concentrations correlated with reduced response potential. This review examines the current evidence evaluating individualized ovarian stimulation protocols using AMH concentration as a predictive marker of ovarian response. The rationale behind why specific treatment protocols based on individual response potential may be more suitable is also discussed. Based on current evidence, it appears that the use of AMH serum concentrations to predict ovarian response and optimize treatment strategies is a promising approach for improving pregnancy outcomes in women undergoing ovarian stimulation. However, prospective randomized controlled trials evaluating this approach are needed before any firm conclusions can be drawn.

The nutritional pattern of frailty - Proceedings from the 5th Italian Congress of Endocrinology of Aging, Parma, Italy, 27-28 March 2009.

Frailty has become one of the 'hot spots' in geriatric research. Frailty has multifactorial origin, and is regarded as a fundamental risk factor for deteriorating health status and disability in elderly people and is highly prevalent in the population above the age 65. It is estimates that prevalence rates up to 27% and pre-frailty rates up to 50% qualify for the term "epidemic". Although the role of nutritional deficiency in the development of age-related frailty was suggested long ago, research conducted in this area is relatively recent. The critical role of micronutrients in this context suggests the need to improve the quality of food eaten by elderly people whereas quantity of food appears to be less relevant. This review summarizes the recent literature on the nutritional pathways to frailty with particular focus on the effect of energy, protein and micronutrients.

Endocrinology of the aging male.

Despite enormous medical progress during the past few decades, the last years of life are still accompanied by increasing ill health and disability. The ability to maintain active and independent living for as long as possible is a crucial factor for ageing healthily and with dignity. The most important and drastic gender differences in aging are related to the reproductive organs. In distinction to the course of reproductive ageing in women, with the rapid decline in sex hormones expressed by the cessation of menses, men experience a slow and continuous decline. This decline in endocrine function involves: a decrease of testosterone, dehydro epiandrosterone (DHEA), oestrogens, thyroid stimulating hormone (TSH), growth hormone (GH), IGF1, and melatonin. The decrease of sex hormones is concomitant with a temporary increase of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In addition sex hormone binding globulins (SHBG) increase with age resulting in further lowering the concentrations of free biologically active androgens. These hormonal changes are directly or indirectly associated with changes in body constitution, fat distribution (visceral obesity), muscle weakness, osteopenia, osteoporosis, urinary incontinence, loss of cognitive functioning, reduction in well being, depression, as well as sexual dysfunction. The laboratory and clinical findings of partial endocrine deficiencies in the aging male will be described and discussed in detail. With the prolongation of life expectancy both women and men today live 1/3 of their life with endocrine deficiencies. Interventions such as hormone replacement therapy may alleviate the debilitating conditions of secondary partial endocrine deficiencies by preventing the preventable and delaying the inevitable.

Rat testis mitochondrial adenylate cyclase. Partial purification and characterization.

1. Adenylate cyclase (EC 4.6.1.1) from rat testis mitochondria has been solubilized by treatment with the non-ionic detergent Lubrol PX. The soluble enzyme was further purified by DEAE-cellulose chromatography. 2. The specific activity of the adenylate cyclase eluted from the DEAE-cellulose column was found to be four times higher than that of an intact mitochondrial preparation. At this step the enzyme shows a sedimentation coefficient of 4.2 S and a diffusion coefficient (D) of 3.12 - 10- minus 7 cm-2/sec. 3. Solubilization of the adenylate cyclase resulted in loss of responsiveness to gonadotrophic hormones. Addition of phosphatidylserine to the soluble preparation partially restored the activation of adenylate cyclase by human chorionic gonadotrophin. 4. The results of this study suggest that the activity of the adenylate cyclase may be dependent on the membrane-bound phospholipids and that the enzyme attached to the mitochondrial membranes has some properties which are similar to the adenylate cyclase found to be associated with other membrane systems of the cell.?

Differences in the association of the progesterone receptor ligated by antiprogestin RU38486 or progestin ORG 2058 to chromatin components.

We assessed the hypothesis that due to variations in the conformation of the progesterone receptor induced by the antiprogestin RU38486 compared to the progestin ORG 2058, differences may result in the association of the receptor with some of the chromatin components. The physical properties of the receptor-bound chromatin fragments released by micrococcal nuclease digestion were characterized by sucrose gradient sedimentation and by gel filtration on Agarose A-1.5m or Agarose A-5m columns. The nuclear fraction was isolated from T47D cells previously exposed to 0.1 microM [3H]RU38486 or 0.1 microM [3H]ORG 2058. Micrococcal nuclease digestion solubilized two receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only one receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking with the cross-linker 2-iminothiolane of the micrococcal nuclease solubilized receptor forms resulted in 6.7-S and 4.4-S forms sedimenting on 0.4 M KCl gradients for the antiprogestin and progestin ligated receptors, respectively. Stokes radii of 7.3 nm and 6.4 nm were determined by gel filtration in 0.4 M KCl for the 6.7-S and the 4.4-S receptor forms, respectively. Using the sedimentation coefficient and the Stokes radius, molecular weights of 202,000 and 116,000 were calculated for the antiprogestin and progestin ligated receptors. We conclude that the micrococcal nuclease solubilized antiprogestin ligated receptor is associated with additional or different chromatin components compared to the progestin bound receptor.

PIP: The hypothesis that variations in the conformation of the progesterone receptor induced by the antiprogesterone RU38486 compared to the progestin ORG2058 may cause differences in the association of the receptor with some of the chromatin components was investigated. The approach to analyzing the functional organization of the receptor in the chromatin was to study the receptor-bound fragments released by nuclear digestion. The physical properties of these receptor-bound chromatin fragments were characterized by sucrose gradient sedimentation and gel filtration. Micrococcal nuclease digestion solubilized 2 receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only 1 receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking resulted in 6.7 S forms for the antiprogestin receptors and 4.4 S forms for the progestin ligated receptors. Use of the sedimentation coefficient and the Stokes radius yielded molecular weights of 202,000 and 116,000 for the antiprogestin and progestin ligated receptors, respectively. Overall, these kinetic studies were unable to explain the variation in responses of the target cell to agonist or antagonist ligands. It is postulated that the antagonist exerts a subtle, but important, conformational change in the receptor, which alters the receptor's ability to associate with some chromatin components and thus affects the normal events in gene expression.

A cytosolic protein tyrosine kinase activity is induced by follicle stimulating hormone and insulin like growth factor-I in human granulosa cells.

Follicle stimulating hormone (FSH) induces estradiol (E2) production in rat, porcine and human granulosa cells with a concomitant increase in cAMP. In human granulosa cells insulin like growth factor-I (IGF-I) induces E2 production without cAMP accumulation. In the current study we report that IGF-I and FSH effects on aromatase activity both involve activation of a cytosolic soluble protein tyrosine kinase (CytPTK). This FSH and IGF-I stimulated CytPTK activity was blocked by AG-82 (a tyrosine kinase inhibitor) and by staurosporine (STS) (a non specific protein kinase inhibitor) at concentrations which inhibited E2 production. These new findings strengthen the concept of fail-safe mechanism in E2 production in human granulosa cells by an involvement of tyrosine kinase(s) activity downstream of cAMP formation and protein kinase A (PKA) activation.

Thyroxine concentration in human milk.

Thyroxine (T4) concentrations were measured by RIA in 37 milk samples from 19 healthy eutryroid mothers, obtained between 3-165 days postpartum. The mean milk T4 concentration in the first week postpartum was 0.38 +/- 0.07 microgram/100 ml (mean +/- SEM). The mean T4 concentrations between 8 and 48 days rose to 4.27 +/- 0.50 microgram/100 ml (mean +/- SEM), and decreased to 1.11 +/- 0.25 microgram/100 ml (mean +/- SEM) after 50 days postpartum. The data suggest that human milk can provide a significant exogenous source of T4 to the infant. In the hypothyroid infant, the amount of T4 in human milk may delay clinical recognition of the disease. Although this exogenous source of T4 may alleviate the disease, it is insufficient to prevent the detrimetal effects of hypothyroidism.

Hypothalamic-pituitary function in patients with prolonged coma.

In eight patients (six males and two females) hospitalized for prolonged coma (164-1320 days; five patients were in traumatic coma, two after cardiac arrest during surgery, and one after a venomous scorpion sting), diurnal variation of cortisol secretion, human (L) GH, PRL, TSH secretion as well as the release of the pituitary hormones after TRH and L-dopa stimulation were assessed. Serum T4 cortisol, hGH, hPRL, and hTSH levels were normal. The cortisol diurnal variation was preserved in six patients. In seven of eight patients, the hPRL and hTSH responses to TRH test were normal and in five patients, there was a rise of more than 150% in serum hGH concentrations. After L-dopa administration, five patients responded with a rise in hGH. In three patients, there was no response of hGH to TRH or L-dopa. Five patients responded with significant reduction in the serum hPRL concentration after L-dopa, and in three patients, no change was observed. It was concluded that patients with prolonged coma have variable hypothalamic pituitary function. They preserve the cortisol diurnal variation and the hypothalamic-pituitary-thyroid axis as well as PRL secretion; however, hGH secretion may respond abnormally to various stimuli.

Estradiol binding to nuclear receptors in human breast cancer tissue (MCF-7 cell line) and in dimethylbenz(a)anthracene-induced mammary carcinoma.

PIP: The presence or absence of estrogen receptors in the nuclei of human breast tumor may be a useful tool in determining whether the tumor will or will not respond to endocrine therapy. This paper describes an assay which measures both unoccupied and occupied nuclear receptors in human breast cancer tumors. The assay was predicated on the fact that at low salt concentration, the nuclear receptor is bound to chromatin particles and can be separated from the soluble components containing proteolytic acitivity. Nuclear estradiol receptors were measured in human breast cancer tissue (MCF-7 cell line) and in DMBA (dimethylbenz(a)anthracene-induced rat mammary carcinomas) tumors. Complete translocation of the cytoplasmic receptor in the MCF-7 cells was observed compared to only 35-50% of the cytoplasmic receptors seen in the nucleus of the DMBA tumor after estradiol injection. The study also showed 6 pmol/mg DNA for total unoccupied nuclei and cytoplasmic estrogen receptors, and 25% of it in the nucleus; this finding differed from Zava et al's finding of 2 pmol/mg DNA and 75% in the nucleus, probably because of differing methodology or use of a later passage of cell line. 29 out of the 34 tumors with cytoplasmic receptors were found to contain unoccupied nuclear receptors, indicating that free nuclear receptors are not exceptions. The assay used in this study is currently being used to determine the translocative ability of the cytoplasmic receptors in human breast carcinomas.^ieng

Reduction of follicle-stimulating hormone (FSH) secretion in FSH-producing pituitary adenoma by bromocriptine.

An FSH-secreting pituitary adenoma was demonstrated in a 32-yr-old man who presented with unilateral optic atrophy without any clinical or laboratory evidence of hypogonadism. Semen analysis was normal, although basal FSH levels were markedly elevated (greater than 80 mIU/ml). He had normal plasma LH levels and no other detectable endocrine abnormalities. Administration of GnRH elicited delayed and sustained FSH and brisk LH responses. Administration of TRH resulted in TSH and PRL responses and unexpected FSH and LH elevations. Two surgical operations resulted in temporary reduction of plasma FSH levels, but it increased later concomitant with CT demonstration of tumor growth. After pituitary irradiation, no reduction in FSH levels occurred. A single dose of 5 mg bromocriptine elicited a significant reduction in FSH levels from 137 to 64 mIU/ml. Long term treatment with 15 mg/day bromocriptine resulted in further reduction of FSH level, to 36.4 mIU/ml, without any change in tumor size. This finding implies that bromocriptine could be an adjunctive therapy or an alternative to other modes of treatment in patients with these rare tumors.

Unoccupied nuclear receptors for estrogen in human endometrial tissue.

PIP: The nuclear content of estrogen-binding sites in normal and pathological endometrial tissue was studied. Extracts of cell nuclei from normal, nonmalignant, and cancerous human endometrial tissues were used, and at 0 degrees centigrade were found to contain material capable of binding estradiol in vitro. Only binding sites unoccupied by endogenous estrogen were determined. Estrogenic receptor (ER) character of this binding was demonstrated by 3 findings: 1) high affinity of binding to estradiol; 2) specificity of binding, competition by diethylstilbestrol and estriol for estradiol binding, and absence of competition by cortisol, progesterone, and 5 alpha-dihydrotestosterone; and 3) sedimentation constant at about 4S in sucrose density gradient. Next, occupied receptors were determined at 30 degrees centigrade. Both occupied and unoccupied receptors were measured by a single saturating dose of 7.5 nM tritiated estradiol with or without a 100-fold excess of diethylstilbestrol to estimate the amount of nonspecific binding. All specimens assayed had unoccupied nuclear receptors. This unoccupied receptor comprised from 9-37% of the total estradiol receptors (cytoplasmic plus nuclear). Such a large percentage of unoccupied nuclear receptors in both normal and pathological endometrial material may indicate that the unoccupied receptor is a necessary product in the normal mechanism of estradiol action.^ieng

Insulin-like growth factor-I and follicle-stimulating hormone suppress insulin-like growth factor binding protein-1 secretion by human granulosa-luteal cells.

Local regulation of insulin-like growth factor binding protein-1 (IGFBP-1) production in the human ovarian follicle was investigated using cultured human granulosa-luteal cells. Both insulin-like growth factor-I (IGF-I) and follicle-stimulating hormone (FSH) exerted a dual effect on granulosa cells: while estradiol (E2) production was increased by both stimulants, the addition of either of the two hormones led to a reduction in IGFBP-1 secretion by more than 50%. Inhibition of IGFBP-1 production in response to IGF-I was dose-dependent,with the highest effect observed at 5 nM IGF-I. A significant correlation was found between the increase in E2 and inhibition of IGFBP-1 secretion in response to IGF-I. These observations may suggest a novel mechanism, at the follicular level, by which FSH and IGF-I amplify the IGF-I effect in the ovarian follicular cells.

Insulin-like growth factor-I (IGF-I) may not be essential for ovarian follicular development: evidence from IGF-I deficiency.

Insulin-like growth factor-I (IGF-I) stimulates growth and differentiation in follicular granulosa cells (GC). To examine whether this effect is prerequisite to human folliculogenesis, a patient with Laron-type dwarfism (IGF-I deficiency secondary to GH receptor abnormality) was examined while undergoing in vitro fertilization treatment. Despite low levels of IGF-I in serum and follicular fluid (less than 3 and less than 2 nmol/L) and very high levels of IGF-I-binding protein, the patient developed normal ovarian follicles. After the administration of GnRH analog (GnRHa) and human menopausal gonadotropin in a dose similar to that used in normovulatory women, estradiol (E2) levels reached above 5000 pmol/L on the day of hCG administration, and mature fertilizable oocytes were retrieved during ovum pickup. The patient's GC E2 production, tested in a primary culture, did not respond to IGF-I after 4 days of incubation, while control cultures showed a significant increase. Only after a priming period of 7 days did IGF-I have a significant effect on E2 production, as observed in the patient's GC culture. This delayed response suggests that the patient's GC were not exposed in vivo to IGF-I. Our data support the view that IGF-I is not required for normal follicular development, but is, rather, a nonessential modulator of FSH action.

Steroid secretion by granulosa cells isolated from a woman with 17 alpha-hydroxylase deficiency.

Steroid synthesis in luteal/granulosa cells harvested after follicular aspiration in a patient with 17 alpha-hydroxylase deficiency was investigated. Follicular stimulation with purified FSH after the suppression of ACTH and gonadotropin secretion by corticoid analogs and superactive GnRH agonist permitted ovum pick-up and the study of steroid synthesis in the obtained granulosa cells. Progesterone synthesis was elevated while 17 alpha-hydroxy progesterone, testosterone, androstenedione, and estrogen production was minimal. Aromatase activity was retained in these cells, as demonstrated by the 100-fold increase in estrogen levels after the addition of androgens. Progesterone levels in follicular fluids were in the normal range, while estrogen, 17 alpha-hydroxy progesterone, and androgen levels were minimal. These results provide in vitro evidence of 17 alpha-hydroxylase deficiency in the granulosa cells of this patient.

In vitro fertilization and primary embryonic cleavage are possible in 17 alpha-hydroxylase deficiency despite extremely low intrafollicular 17 beta-estradiol.

Congenital adrenal hyperplasia due to 17 alpha-hydroxylase deficiency in genotypic females is characterized by primary amenorrhea and the absence of sexual maturation due to inadequate biosynthesis of ovarian androgens and estrogens. We induced ovarian follicular development in a woman with this syndrome. Ovum pick-up, in vitro fertilization, and primary embryonic development were achieved despite undetectable plasma estradiol and extremely low ovarian androgen concentrations and minute concentrations of these hormones in the ovarian follicular fluid.

Enhanced response of plasma aldosterone to metoclopramide in essential hypertension.

Aldosterone responses to posture and the dopamine antagonist, metoclopramide, were studied in seven normotensive controls and 12 patients with essential hypertension. Both groups had similar basal supine plasma renin activity and aldosterone levels. Aldosterone levels of the hypertensive patients were greater than those of the controls 10 min after assuming an upright posture but indistinguishable at 120 min. Metoclopramide induced a peak fourfold increase above basal aldosterone levels in the hypertensive group as compared to a peak twofold increase observed in the normotensive controls. Mean 120-min integrated aldosterone response area for the hypertensives (237 +/- 44 10(-10) mol min/l) was greater (P less than 0.05) than that for normotensive subjects (106 +/- 32 10(-10) mol min/l). Simultaneous cortisol, plasma renin activity, and serum potassium levels were unaffected by metoclopramide. It is concluded that dopaminergic modulation of aldosterone secretion may be altered in essential hypertension.

Effect of the inhibitory material from male genital tract on natural killing activities.

A male inhibitory material (HuSePl-Frl) was separated from normal human seminal plasma by gel filtration on Sephadex G-100. This material has high avidity for cell membranes of human red blood cells (RBC). In vitro, PHA-M-induced blast transformation of human lymphocytes is inhibited by small quantities of HuSePl-Frl. This inhibition was also evident when RBC coated with HuSePl-Frl is added to mitogen stimulated lymphocytes in cultures. Small quantities of HuSePl-Frl are inhibitory to natural killer (NK) cell activation. The modulating role of the HuSePl-Frl in preventing the spermatozoal rejection during fertilization is discussed.

Unoccupied binding sites for oestradiol in nuclei from human breast carcinomatous tissue.

The binding of oestradiol to a nuclear fraction extracted from human breast carcinomatous tissue was demonstrated. The material, which was extracted with KCl, sedimented at 3--4S and bound oestradiol with high affinity (dissociation constant approximately 2 X 10(-10) mol/l). Oestriol, diethylstilboestrol and 5 alpha-dihydrotestosterone (100-fold excesses) competed with [3H]oestradiol for the binding sites (binding inhibited by 89 +/- 8 (S.D.), 92 +/- 6 and 57 +/- 8% respectively), whereas progesterone and cortisol (100-fold excesses) did not (binding suppressed by 5 +/- 5 and 2 +/- 3% respectively). Similar competition patterns were found for cytoplasmic material which bound oestradiol. The binding occurred at 4 degrees C and was therefore considered to be a measure of the amount of binding material unoccupied by endogenous oestrogen, Unoccupied binding sites for oestradiol in the nucleus and cytoplasm were measured in 35 samples of breast carcinomatous tissue using sucrose gradient centrifugation. In 17 out of 35 tumorus, unoccupied nuclear and cytoplasmic 8S and 4S binding sites could be detected. Three out of 35 tumours contained unoccupied nuclear binding sites and 4S cytoplasmic binding sites. Nuclear binding sites only were found in two out of 35 tumours. Unoccupied nuclear binding sites were not detected in 13 out of 35 tumours and ten of these tumours also did not contain unoccupied cytoplasmic binding sites.

PIP: This study examines unoccupied binding sites for estradiol by using carcinomatous tissues of human breast. Estradiol, estriol, diethylstilbestrol and dihydrotestosterone demonstrated a higher affinity for binding sites, while progesterone and cortisol did not. Similar patterns were found for cytoplasmic material, and the binding occurred at 40C. In addition the quantities of unoccupied binding sites for estradiol in nuclei and in cytoplasm from 35 samples of human breast carcinomatous tissue were measured. Nuclear binding sites only were found in 2 out of 35 tumors, while unoccupied nuclear binding sites were not detected in 13 out of 35 tumors; 10 of these 13 did not contain unoccupied cytoplasmatic binding sites.

Synergistic effect of growth hormone and gonadotropins in achieving conception in "clonidine-negative" patients with unexplained infertility.

Based on preliminary reports by others and by us of a potentiating effect of growth hormone (GH) on human menopausal gonadotropin (hMG)-induced ovulation, a study using a randomized, prospective, cross-over protocol between GH + hMG/human chorionic gonadotropin (hCG) and hMG/hCG was undertaken. The study included patients with long-standing (2-11 years) unexplained infertility with a negative or reduced GH response to clonidine (up to 150 micrograms of clonidine orally). The first cycle was randomly assigned between GH/hMG/hCG (study cycle) and hMG/hCG alone (control cycle), and after an interval cycle the patient's treatment was crossed over. All patients who completed the study had previously undergone 1-20 attempts at ovulation induction for in vitro fertilization (IVF) and 5-40 cycles of ovulation induction for in vivo fertilization at three different medical centers. Three patients conceived on the combined GH/hMG cycle, with diminution in the hMG consumption needed for ovulation induction in the study cycles. Another patient with long-standing mechanical infertility underwent 11 abortive attempts at ovulation induction with hMG for IVF but has never achieved egg retrieval. On the GH/hMG/hCG ovulation induction cycle, three mature ova were retrieved as opposed to no response and cancellation of the "hMG only" cycle. Another patient with 11 years of primary infertility who had undergone 21 previous attempts at ovulation induction and had reached follicular aspiration in only three of those cycles conceived spontaneously on the first cycle after the GH/hMG/hCG IVF/ET cycle. All four pregnancies that have been achieved by now in seven GH/hMG-treated patients ended in cesarean deliveries of four normal male neonates. No correlation was found between the follicular fluid levels of insulin-like growth factor I (IGF-I) and the fertilization rate in vitro. The peripheral IGF-I levels were significantly higher during the follicular phase of the study cycles than during the respective stage of the control cycles or the luteal phase of either cycle. A study of serum GH-binding protein (GH-BP) levels revealed gradual increases in the late follicular phase, in the luteal phase, and in early pregnancy. On the basis of this study and in keeping with earlier reports, we conclude that the addition of GH to hMG/hCG may serve as a contributory adjunct in selected patients. However, in contrast to others who could not find a correlation between the response to acute tests for GH release and the ovarian response to combined treatment, we conclude that the clonidine test can play a discriminatory role in identifying patients who may benefit from this innovative combination.

Conception rate after gonadotropin therapy in hyperprolactinemia and normoprolactinemia.

The cumulative pregnancy rate after gonadotropin treatment was evaluated in 63 hyperprolactinemic and 242 normoprolactinemic women. All pregnancies in the hyperprolactinemic patients were achieved within four treatment cycles; the cumulative pregnancy was 62% as compared with 29% in normoprolactinemics. The same results were obtained when patients were divided according to endogenous estrogenic activity. These results imply that in bromocriptine failures there is no need to lower prolactin levels to achieve pregnancy with gonadotropins.