Marcelo C. Batista

Apparent mineralocorticoid excess in a Brazilian kindred: hypertension in the heterozygote state

Background Apparent mineralocorticoid excess (AME) is a cause of low-renin, low-aldosterone hypertension in which cortisol acts as a mineralocorticoid. The condition reflects an inability to inactivate cortisol to cortisone due to defective activity of the type 2 isozyme of 11b-hydroxysteroid dehydrogenase (11β-HSD2). Homozygous mutations in 11b-HSD2 gene in patients with AME have been described. A 7-year-old Brazilian girl had previously been found to have AME. Her father recently presented with mineralocorticoid hypertension at age 38 years. Objective To describe the clinical details, to perform steroid analyses and to assess the molecular basis for the hypertension in this kindred. Methods The 11βHSD2 gene was amplified from genomic DNA by the polymerase chain reaction and sequenced by direct chain-termination sequencing on an automatic DNA sequencer. The sequencing results were validated by restriction-site polymorphism. The mutant 11β-HSD2 protein was expressed in Chinese hamster ovary polyoma cells and enzymatic activity was assessed by metabolizing cortisol in vitro. Results Sequence analysis of genomic DNA revealed a novel C1061T point mutation in exon V of the human 11β-HSD2 gene, resulting in an amino acid substitution of alanine by valine at codon 328 of the enzyme protein (A328V). Expression studies confirmed that the mutant protein was devoid of 11β-HSD2 activity. A HhaI restriction-site polymorphism confirmed that the proband was homozygous for the mutation whereas both parents were heterozygotes. The father of the proband had hypertension, a normal serum potassium level, suppressed plasma renin activity and plasma aldosterone level and a moderately elevated urinary cortisol: cortisone metabolite ratio. Conclusions AME in this kindred is caused by a novel mutation in the 11β-HSD2 gene. Detection of hypokalaemia, at least in this kindred, is an insensitive screening test for mineralocorticoid-based hypertension. In contrast to results from previously investigated kindreds, we have demonstrated that this kindred has an abnormal phenotype in the heterozygote state. Further studies are now required in order to evaluate the role of 11β-HSD2 activity in the pathophysiology of ‘essential’ hypertension.

The antiprogestin RU486 delays the midcycle gonadotropin surge and ovulation in gonadotropin-releasing hormone-induced cycles †

OBJECTIVE To investigate whether the antiprogestin RU486 acts primarily on the hypothalamus to delay the midcycle gonadotropin surge and thus gain insight into the site(s) of action of P in the control of ovulation. DESIGN Prospective, crossover, single-blinded clinical study. SETTING Outpatient clinic in an academic research environment. PATIENTS Women with hypothalamic amenorrhea. INTERVENTIONS RU486 or a placebo was given orally at a low dose of 1 mg/d for 5 days, starting when the dominant follicle reached 14 to 16 mm, to women with hypothalamic amenorrhea undergoing ovulation induction with GnRH pulses of unvarying frequency and dose. Blood samples and ovarian ultrasounds were obtained daily in the late follicular phase and every 3 to 4 days in the remainder of the cycle. MAIN OUTCOME MEASURES Follicular diameter and plasma levels of LH, FSH, E2, and P. RESULTS RU486 consistently delayed the timing of the midcycle gonadotropin surge and ovulation. Gonadotropin and steroid levels were suppressed during RU486 treatment, but follicular growth progressed normally in most patients. CONCLUSIONS RU486 does not act primarily on the hypothalamus to delay ovulation. Rather, this compound appears to antagonize P at the pituitary level to suppress gonadotropin and steroid hormone secretion. P may thus act on the pituitary, independent of any hypothalamic effects, to regulate the timing of the midcycle gonadotropin surge and ovulation.

Clinical features of women with resistance to luteinizing hormone

To review clinical and hormonal characteristics of new and published females with LH resistance.

Ovarian resistance to luteinizing hormone: a novel cause of amenorrhea and infertility.

OBJECTIVE To report the clinical, hormonal, and histopathological features of a woman with ovarian resistance to LH. DESIGN Clinical study. SETTING University hospital. PATIENT(S) A woman with amenorrhea, sister of a patient with male pseudohermaphroditism due to Leydig cell hypoplasia. INTERVENTION(S) Blood drawing before and after GnRH stimulation and also after dexamethasone and hCG administration, pelvic ultrasound, and ovarian biopsy. MAIN OUTCOME MEASURE(S) Karyotype, gonadotropin and steroid measurements, follicular diameter, ovarian histology, and sequencing of the LH receptor gene. RESULT(S) Patient had normal female external genitalia, normal breast development at puberty, rare episodes of vaginal bleeding, and infertility. The karyotype was 46,XX. She had elevated serum LH levels, whereas E2 and P concentrations were in the range seen in the early follicular phase. Pelvic ultrasound revealed a slightly hypoplastic uterus and enlarged polycystic ovaries. A normal follicular reserve for age, antral follicles, and absence of corpora lutea or albicans were observed on ovarian biopsy. Exon 11 of the LH receptor gene had a normal sequence. CONCLUSION(S) In our patient with ovarian resistance to LH, FSH stimulated follicular development until the preovulatory stage, but E2 levels remained in the early follicular phase range, still sufficient for normal pubertal feminization. Apparently, LH is necessary for ovulation and corpus luteum formation.

Daily administration of the progesterone antagonist RU 486 prevents implantation in the cycling guinea pig

Since progesterone is required to prepare the endometrium for implantation of an embryo, a progesterone antagonist may inhibit nidation and thus prevent pregnancy. We addressed this possibility in the guinea pig, the small laboratory animal whose reproductive physiology most resembles that of women. Daily administration of the antiprogestin RU 486 (0, 1, 2, or 3 mg/kg, subcutaneously) for 9 days after mating inhibited implantation in a dose-dependent fashion. When this compound was given daily throughout the estrous cycle, cyclic vaginal changes, ovulation, and mating were suppressed in up to 17%, 28%, and 55% of animals, respectively. Two of seven mated female animals receiving RU 486, 1 mg/kg/day, had implantation sites. Nidation was completely blocked at higher doses. Thus daily antiprogestin administration prevented pregnancy in sexually active, normally cycling guinea pigs. A similar strategy using a daily antinidatory dose of an antiprogestin may offer a novel approach to human fertility control.

Effects of long-term storage of filter paper blood samples on neonatal thyroid stimulating hormone, thyroxin and 17-alpha-hydroxyprogesterone measurements.

Objectives Clinical laboratories store filter paper samples used in neonatal screening for various periods of time after performing hormonal measurements. However, due to lack of data concerning specimen stability, it is unclear for how long these samples should be stored. The objective of this study is to determine the stability and reproducibility of thyroid-stimulating hormone (TSH), thyroxine (T4) and 17-hydroxyprogesterone (17-OHP) measurements in filter paper blood samples stored for up to 60 months. Methods Two hundred and twenty-eight blood samples, drawn between the second and the fourth day of life, were divided into seven distinct groups and kept at 4–8°C for one day or 2, 12, 24, 36, 48 or 60 months after basal hormonal measurements. In each group, TSH, T4 and 17-OHP levels were initially assayed 24–48 hours after collection (basal) and repeated once at the end of storage timing. All the measurements were performed by time-resolved fluorometry (1235 AutoDELFIA, Wallac Oy, Turku, Finland). Repeated and basal levels of each hormone were compared within the same group by Students paired t-test. Differences were considered significant at P < 0.05. Results Compared with basal measurements, TSH and T4 levels declined significantly only when these hormones were re-assayed at 48 or 60 months of sample storage. In contrast, 17-OHP concentrations decreased earlier, starting at 24 months and continuing throughout the remaining period. Conclusion Our data suggest that neonatal screening of filter paper samples kept at 4–8°C are reliable for repeating the hormonal measurements when specimens are stored for up to one year, in the case of 17-OHP, or three years, in the case of T4 and TSH.

A prospective controlled study of luteal and endometrial abnormalities in an infertile population.

OBJECTIVE To investigate whether luteal and endometrial abnormalities occur more frequently in an infertile population and thus contribute to infertility. DESIGN Prospective controlled clinical study. SETTING Outpatient clinic in an academic research institution. PARTICIPANTS Thirty-three fertile controls and 31 infertile women without ovulatory disorders, tubal disease, or male factors. INTERVENTIONS All women underwent an endometrial biopsy 9 days after the LH surge followed by an IM injection of 5,000 IU hCG. Blood samples were drawn immediately before hCG administration for serum P and placental protein 14 (PP14) measurements, at 6 hours after hCG stimulation for serum P concentrations, and on day 5 after hCG administration for serum PP14 levels. MAIN OUTCOME MEASURES Histologic dating of the endometrium and serum P and PP14 measurements. RESULTS Abnormal endometrial biopsies occurred more frequently in infertile (43%) than in fertile women (9%). Except for one case, these specimens were not associated with low hCG-stimulated P levels. Serum PP14 measurements varied widely and did not discriminate subjects with abnormal endometrial development. CONCLUSIONS Disruption of endometrial maturation without a concomitant defect of the corpus luteum occurs more frequently in an infertile population and thus may contribute to infertility.

Characterization of the normal progesterone and placental protein 14 responses to human chorionic gonadotropin stimulation in the luteal phase

OBJECTIVE To examine whether midluteal phase administration of the luteotrophic hormone hCG can result in higher and more stable serum levels than random sampling of P and placental protein 14 (PP14). DESIGN Prospective controlled clinical study. SETTING Normal human volunteers in an academic research environment. PARTICIPANTS Twenty-six fertile, regularly cycling women. INTERVENTIONS Blood samples were drawn at 0, 3, 6, 9, 12, 18, and 24 hours and then daily for the next 6 days, after a single IM injection of 5,000 IU hCG or saline given on day 5, 7, or 9 after the LH surge, as detected by rapid plasma assays. MAIN OUTCOME MEASURES Serum P and PP14 measurements. RESULTS Peak P and PP14 concentrations occurred at 6 hours and 5 days, respectively, after hCG stimulation on luteal phase day 9. Progesterone but not PP14 levels were significantly higher and less variable after hCG than after saline administration on this day. Progesterone responses exceeded 11.0 ng/mL (35.0 nmol/L) in all women, suggesting that this represents the cutoff limit for normal luteal function. Because PP14 responses were highly variable and inconsistent, it was not possible to determine a threshold for normal endometrial function. CONCLUSIONS Midluteal phase administration of hCG in normal women induces consistent serum P levels > 11.0 ng/mL (35.0 nmol/L) but highly variable PP14 responses.

Comparative analysis of progesterone and placental protein 14 measurements in the evaluation of luteal function

OBJECTIVE Our purpose was to investigate the diagnostic accuracy of single or summed measurements of progesterone and placental protein 14, a progestin-dependent endometrial glycoprotein, in the evaluation of luteal function. STUDY DESIGN Forty-five healthy women had daily blood measurements of luteinizing hormone, progesterone, and placental protein 14 during one menstrual cycle. RESULTS Thirty-nine women had normal and six had deficient luteal function on the basis of serial progesterone determinations. Luteal insufficiency was not accurately diagnosed by single progesterone or placental protein 14 values or by integrated placental protein 14 measurements. In contrast, the condition was correctly identified in all but one cycle when the sum of progesterone on days 4 and 7 was < 49 nmol/L (15.4 ng/ml). A poor correlation was found between peak or integrated measurements of progesterone and placental protein 14. CONCLUSION Measurement of serum progesterone, but not placental protein 14, on 2 days of the midluteal phase provides a convenient and reliable test of luteal function.

Diagnostic Value of Basal and Stimulated Levels of Gonadotropins Measured By An Immunofluorometric Assay (Ifma) in the Differential Diagnosis of Precocious Puberty. 20

The new immunofluorometric kits such as DELFIA (Wallac, Turku, Finland) have an improved sensitivity and may allow a better discrimination between patients with gonadotropin dependent (GDPP) and independent (IGPP) precocious puberty. Normal prepubertal girls had gonadotropin levels measured by DELFIA kits under basal conditions (n = 20) and after stimulation with 100 μg iv GnRH (n = 10). Basal LH and FSH levels were 0.7 U/L) and FSH (> 3.4 U/L) may establish the diagnosis of GDPP, precluding the use of GnRH stimulation tests. In contrast, basal prepubertal concentrations of LH and FSH may occur in both GDPP and IGPP, indicating the need to perform GnRH stimulation tests.