DAX-1 and DAX-1A expression in human testicular tissues with primary spermatogenic failure.

DAX-1 [dosage-sensitive sex reversal-adrenal hypoplasia congenital (AHC) critical region on the X chromosome gene 1; NR0B1] is an orphan nuclear receptor that acts as a transcriptional repressor in adrenal/gonadal development, steroidogenesis and probably spermatogenesis. An alternatively spliced form called DAX-1A (NR0B1A) has been described in several tissues including the testis, and in vitro studies have shown an inhibitory effect on DAX-1 transcriptional function. We aimed to study the mRNA and protein expression of DAX-1 in testicular tissues of 65 men with primary spermatogenic failure [complete Sertoli cell only syndrome (SCOS), focal SCOS, maturation arrest and mixed atrophy] compared with 33 controls with normal spermatogenesis. As a novel finding, we observed intense immunostaining, not only in the nucleus of Sertoli cells, but also in pachytene spermatocytes and round spermatids. The quantitative mRNA expression of DAX-1 and DAX-1A was similar between cases and controls and was not associated with the levels of gonadotrophins and steroids. Moreover, DAX-I transcript expression level was ∼750-fold higher than DAX-1A, and there was a strong positive correlation between them (r = 0.52; P< 0.001). We conclude that, in addition to Sertoli cells, DAX-1/DAX-1A is expressed in germ cells from spermatogonia to round spermatids. Besides, the similar mRNA expression of DAX-I and DAX-IA in testicular tissues from cases and controls does not support the involvement of DAX-1 in the etiology of primary spermatogenic failure. Finally, the low level of expression of the alternative transcriptional variant DAX-1A would not support its putative inhibitory function in vivo.

P450-aromatase activity and expression in human testicular tissues with severe spermatogenic failure.

There is evidence that impaired spermatogenesis is associated with an imbalance in the oestradiol/testosterone ratio and with Leydig cell (LC) dysfunction. In testis, P450-aromatase, encoded by CYP19, is responsible for the conversion of testosterone to oestradiol. The aims of this study were to quantify CYP19 mRNA expression, aromatase activity and protein localization, and to measure the oestradiol to testosterone ratio in testicular tissues of men with spermatogenic impairment. Twenty-four men with complete Sertoli cell-only syndrome (SCOS), 14 with focal SCOS, 14 with maturation arrest (MA), 8 with mixed atrophy and 30 controls with normal spermatogenesis were subjected to testicular biopsy. All subjects underwent a physical examination, cytogenetic and serum hormonal studies. Testicular CYP19 mRNA was quantified using real time RT-PCR. Testicular aromatase activity was measured using the (3)H(2)0 assay and protein expression was evaluated using immunohistochemistry. In cases, serum testosterone and oestradiol were normal, but the testosterone/LH ratio was lower compared with controls (p < 0.05). Aromatase was localized in the Leydig, Sertoli and germ cells of all tissues, although stronger intensity was observed in LC. Aromatase mRNA and activity were not altered in cases and correlated positively with LC number (r = 0.516 and r = 0.369; p < 0.008). The intratesticular oestradiol/testosterone ratio was elevated (p = 0.005) in complete SCOS patients compared with controls. In conclusion, testicular aromatase seems to be normal in most subjects with impaired spermatogenesis. However, an altered intratesticular oestradiol/testosterone ratio in some patients with complete SCOS suggests that aromatase is increased, which might contribute to Leydig cell dysfunction.

The G11778A LHON mutation does not enhance ethambutol cytotoxicity in a cybrid model.

UNLABELLED: Leber's hereditary optic neuropathy (LHON) is a maternally inherited mitochondrial disorder, leading to a selective loss of retinal ganglion cells (RGC) and degeneration of the optic nerve, which results in severe visual impairment or even blindness. The primary causes are point mutations of the mitochondrial DNA (mtDNA), associated with aminoacid exchanges in complex I of the electron transport chain (ETC), which are thought to disturb oxidative ATP generation in the mitochondria. The major side effect of the antibiotic ethambutol, commonly used in tuberculosis therapy, is a retinopathy, which may lead to selective RGC loss, if not detected in an early stage. Moreover, LHON was reported to be elicited by ethambutol in some mutation carriers. OBJECTIVE: The present study intended to measure a possible synergism between mitochondrial dysfunction, caused by the most common LHON mutation (G11778A) and caused by ethambutol, which may lead to a higher cytotoxicity of the drug in LHON cells. MATERIAL: An NT2/D1 teratoma-derived LHON cybrid line and the parental cells. METHOD: Determination of ethambutol toxicity in both lines, using a microtiter tetrazolium assay, luminometric measurement of ATP/ADP ratios and determination of mtDNA copy numbers by Real-time PCR. RESULTS: Short-term ethambutol toxicity occurred only at micromolar concentrations, far beyond the estimated plasma peak concentrations of patients under antibiotic therapy. No significant difference occurred between both cell lines. The ATP/ADP ratios in the cybrids were surprisingly low, but showed no correlation with the mutational status of drug-treated cells. The mtDNA copy number of treated LHON and parental cells did not differ significantly. CONCLUSIONS: Ethambutol shows no synergism with the most common primary LHON mutation with respect to mitochondrial energy production or mtDNA replication in cybrid cells, although the issue of ATP decline should be further addressed in neuronally differentiated cybrids with complete OXPHOS dependency.

Quantification of DDX3Y, RBMY1, DAZ and TSPY mRNAs in testes of patients with severe impairment of spermatogenesis.

Y chromosome microdeletion is the most important genetic cause of impairment of spermatogenesis. Nevertheless, a significant proportion of patients with spermatogenic failure do not have this condition. This study investigated the expression level of AZF genes, DDX3Y (DBY), RBMY1, DAZ and TSPY in testicular tissues of 42 subjects with impaired spermatogenesis compared with 33 with normal spermatogenesis. Histopathological evaluation was performed in all subjects and tissues were classified according to Johnsen Score. Transcript amounts were determined by quantitative-competitive RT-PCR. Patients with complete Sertoli cell-only syndrome (SCOS) did not exhibit RBMY1, DAZ and TSPY gene expression, however, we detected very low expression of DDX3Y transcript. Tissue samples with focal SCOS showed significantly decreased expression of all genes (P < 0.001). Maturation arrest and hypospermatogenesis tissues expressed significantly low levels of DDX3Y testicular transcript (P < 0.001), while the mRNA levels of the other genes were similar to that in tissues from the normal spermatogenesis group. Negative or diminished gene expression of DDX3Y, RBMY1, DAZ and TSPY in tissues samples with SCOS or focal SCOS reflects the absence or the lower number of germ cells, respectively. The finding that the testicular transcript of DDX3Y is significantly decreased in patients with severe spermatogenenic failure, especially in those presenting maturation arrest, suggests an important role of DDX3Y during spermatogenesis.

Nerve growth factor-dependent activation of trkA receptors in the human ovary results in synthesis of follicle-stimulating hormone receptors and estrogen secretion.

CONTEXT: Previous studies showed that nerve growth factor (NGF) induces the expression of functional FSH receptors (FSHR) in preantral follicles of the developing rat ovary. OBJECTIVE: The objective of this study was to determine whether NGF can affect granulosa cell (GC) function in human periovulatory follicles using intact human ovaries and isolated human GCs. PATIENTS AND INTERVENTIONS: Human GCs were obtained from in vitro fertilization patients and normal ovaries from women with elective pelvic surgery for nonovarian indications. RESULTS: In normal ovaries, NGF and trkA (NGF's high-affinity receptor) were detected by immunohistochemistry in GCs of preantral and antral follicles. NGF and trkA are also present in thecal cells of antral follicles. Both freshly collected and cultured GCs contained immunoreactive NGF and trkA in addition to their respective mRNAs. Human GCs respond to NGF with increased estradiol (E(2)) secretion and a reduction in progesterone output. Exposure of human GCs to NGF increased FSHR mRNA content within 18 h of treatment, and this effect was blocked by the trk tyrosine kinase blocker K-252a. Also, cells preexposed to NGF released significantly more E(2) in response to hFSH than cells not pretreated with the neurotropin, showing that the NGF-induced increase in FSHR gene expression results in the formation of functional FSHRs. CONCLUSIONS: These results suggest that one of the functions of NGF in the preovulatory human ovary is to increase the secretion of E(2) while preventing early luteinization via an inhibitory effect on progesterone secretion. NGF stimulates E(2) secretion both directly and by increasing the formation of FSHRs.

Operative fetoscopy via telesurgery.

We describe a case in which telesurgical consultation from Tampa, Florida, USA was used to accomplish operative fetoscopy in Santiago, Chile for the treatment of a twin pregnancy involving an acardiac twin. The procedure was successful and a healthy infant was delivered at 37.5 weeks. Operative fetoscopy, a surgical approach to correct birth defects in utero via combined ultrasound and endoscopy, is only available in a handful of centers worldwide. Telesurgery makes use of telecommunication to allow a surgeon at a primary operating site to consult with another experienced surgeon for complex surgical cases. This case illustrates the potential for ultrasound and telesurgery to expand the horizons of operative fetoscopy.

Hormonal profile, endometrial histology and ovarian ultrasound assessment during 1 year of nomegestrol acetate implant (Uniplant).

The present study assesses the endocrinological, endometrial histology and vaginal ultrasound profiles of nomegestrol acetate subdermal implant users at varying times after insertion. Follicle stimulatory hormone, luteinizing hormone, oestradiol, progesterone, vaginal ultrasound assessment of the ovaries and the histological dating of the endometrium were serially assessed for a period of 50 days immediately after the insertion, and after at 6 months and 12 months of use. The endocrinological results of this prospective observational clinical trial indicated that 75% of the cycles across the study period in Uniplant users were anovulatory, 63% showing development of a persistent non-luteinized follicle. Anovulatory cycles devoid of follicular development were seen primarily in the first months after Uniplant insertion. Ovulatory cycles represented 25% of the Uniplant cycles. Inadequate luteal phase or disregulation of follicular growth was a common feature of ovulatory cycles. In conclusion, these findings suggest that the contraceptive mechanisms of a single nomegestrol acetate subdermal implant involve prevention of follicular growth, development of a persistent non-luteinized follicle, inadequate luteal phase and disruption of the endometrial architecture.

Neuroendocrine mechanism of anovulation in users of contraceptive subdermal implant of nomegestrol acetate (Uniplant).

OBJECTIVE: To evaluate a nomegestrol acetate subdermal contraceptive implant's (Uniplant; Thermex, Monaco) effect on the hypothalamus-pituitary-ovarian axis. DESIGN: A prospective clinical trial. SETTING: San Borja-Arriarán Clinical Hospital, University of Chile, School of Medicine. PATIENT(S): Normally cycling healthy women. INTERVENTION(S): Insertion of Uniplant. MAIN OUTCOME MEASURE(S): Luteinizing hormone pulse and endocrine profiles were assessed before, 48 hours after insertion, and after prolonged use of the implant. RESULT(S): Anovulation was noted in 100% of users in the first month. Seventy percent of subjects demonstrated follicular development with the absence of ovulation and an endocrine profile similar to the follicular phase: (LH pulse/8 hours 6.85 +/- 0.67, LH amplitude 3.54 +/- 0.65 mIU/mL (conversion factor to SI unit, 1.00), and E2 193 +/- 29.4 pg/mL (conversion factor to SI unit, 3.67), whereas 30% demonstrated no follicular activity with an endocrine profile similar to the luteal phase: (LH pulse/8 hours; 3.66 +/- 0.66, LH amplitude 5.76 +/- 1.73 mIU/mL, and E2 67.5 +/- 4 pg/mL. Clinical characteristics, serum gonadotropin concentration, and LH pulse characteristics failed to predict which subjects would initiate or remain devoid of follicular activity. CONCLUSION(S): Uniplant results in anovulation via two mechanisms: hypothalamic suppression in subjects who lack follicular development, and likely suppression of the pituitary LH surge in subjects who initiate follicular activity.

PIP: Findings are reported from a study of a nomegestrol acetate subdermal contraceptive implant's (Uniplant) effect upon the hypothalamus-pituitary-ovarian axis. 10 normally cycling healthy women aged 22-36 years requesting a contraceptive method at San Borja-Arriaran Hospital's family planning clinic participated in the prospective clinical trial. Luteinizing hormone pulse and endocrine profiles were assessed before insertion, 48 hours after insertion, and after prolonged use of the implant. Anovulation was noted in 100% of users in the first month. 70% of subjects demonstrated follicular development with the absence of ovulation and an endocrine profile similar to the follicular phase, while the remaining 30% demonstrated no follicular activity with endocrine profile similar to the luteal phase. Clinical characteristics, serum gonadotropin concentration, and LH pulse characteristics failed to predict which subjects would initiate or remain devoid of follicular activity. Uniplant therefore results in anovulation through hypothalamic suppression in subjects who lack follicular development and the likely suppression of the pituitary LH surge in subjects who initiate follicular activity.

Changes in the menstrual bleeding of users of a subdermal contraceptive implant of nomegestrol acetate (Uniplant) do not influence sexual frequency, sexual desire, or sexual enjoyment.

OBJECTIVE: To evaluate the effect of menstrual changes induced by a nomegestrol acetate subdermal contraceptive implant (Uniplant; Thermex, Bahia, Brazil) on users' sexuality. DESIGN: Prospective observational survey. SETTING: San Borja-Arriarán Hospital, University of Chile, School of Medicine. PATIENT(S): Normally cycling healthy women and their partners. INTERVENTION(S): Structured interview before and during use of the contraceptive. MAIN OUTCOMES MEASURE(S): Sexual frequency, desire, and enjoyment; perception of health; and contraceptive satisfaction. RESULT(S): During the use of the implant more women reported irregular cycles (32% versus 11%) and vaginal spotting (38% versus 19%). Frequency of sexual relations was unchanged (2.3 versus 2.5/wk) but the percent of couples engaging in sexual relations during vaginal spotting increased (28% versus 11%). There was no significant difference in the percent of men or women who reported an increase, or decrease, in perceived sexual desire, sexual enjoyment, or perception of health during the use of Uniplant. CONCLUSION(S): Despite the alterations in menstrual cyclicity and the occurrence of spotting, the use of a contraceptive subdermal implant of nomegestrol acetate did not effect desire for, enjoyment of, or frequency of sexual relations in users.

PIP: Findings are presented from a study evaluating the effect of menstrual changes induced by a nomegestrol acetate subdermal contraceptive implant (Uniplant) on users' sexuality. The Uniplant implant evaluated is manufactured by Thermex of Bahia, Brazil. New subjects enrolled in a phase III clinical trial of the implant at San Borja-Arriaran Hospital, University of Chile, School of Medicine were observed prospectively. The participants were 118 normally cycling healthy women of median age 28 years and 60 male partners of median age 31 years. A structured interview was held before and during use of the contraceptive. Before insertion of the implant, 11% of the women reported irregular menstrual cycles, 19% reported vaginal spotting, and 11% of the couples reported engaging in sexual relations during vaginal spotting. During use of the implant, 32% of the women reported irregular cycles, 38% reported vaginal spotting, and 28% of the couples reported engaging in sexual relations during vaginal spotting. The frequency of sexual relations remained unchanged at 2.3-2.5 acts of coitus per weeks. There was no significant difference in the percentage of men or women who reported an increase or decrease in perceived sexual desire, sexual enjoyment, or perception of health during the use of Uniplant.

[Fetal endoscopic surgery in a case of twin pregnancy complicated by reversed arterial perfusion sequence (TRAP sequence)]. / Cirugia endoscópica fetal en un caso de embarazo gemelar complicado con secuencia de perfusión arterial reversa (secuencia TRAP).

We present the first report in our country of endoscopic fetal surgery in a patient with a twin pregnancy and twin reversal arteral perfusion (TRAP sequence). We performed fetoscopic ligation of the umbilical cord of an acardiac twin at 26 week's gestation.

Donaldson v. Van de Kamp: cryonics, assisted suicide, and the challenges of medical science.

In recent years, advances in medical science have left the legal community with a wide array of social, ethical, and legal problems previously unimaginable. Historically, legislative and judicial responses to these advances lagged behind the rapid pace of such developments. The gap between the scientist's question, "Can we do it?," and the lawyer's question, "Should/may we do it?'" is most evident in the field of cryonics, with its technique of cryonic, or cryogenic, suspension. In cryonic suspension, a legally dead but biologically viable person is preserved at an extremely low temperature until advances in medical science make it possible to revive the person and implement an effective cure. The terminally ill patient who wishes to benefit from such treatment is faced with the dilemma that present life must be ceased with hope of future recovery. As a result, the process challenges our traditional notions of death and the prospects of immortality while raising a host of concomitant legal dilemmas. Some facets of this dilemma are exemplified by Donaldson v. Van de Kamp. In Donaldson, Thomas A. Donaldson sought the declaration of a constitutional right to premortem cryonic suspension of his body and the assistance of others in achieving that state. Donaldson, a forty-six-year-old mathematician and computer software scientist, suffers from a malignant brain tumor that was diagnosed by his physicians in 1988. This tumor is inoperable and continues to grow and invade his brain tissue. Donaldson's condition will gradually deteriorate into a persistent vegetative state and will ultimately result in death. Physicians predict his probable death by August 1993. Donaldson petitioned the California courts, seeking a declaration that he had a constitutional right to achieve cryonic suspension before his natural death. His doctors believe that if Donaldson waits until his natural death to be suspended, future reanimation will be futile because the tumor will have destroyed his brain. In addition, Donaldson's doctors sought an injunction against criminal prosecution for their participation in the suspension, because Donaldson, once suspended, would be considered "dead" under California law. Donaldson and his doctors built their novel argument upon the recent right-to-die cases in which the courts recognized a patient's right to have life-sustaining medical treatment withdrawn. Donaldson argued that his right to privacy and self-determination is paramount to any state interest in maintaining life. Thus, according to Donaldson, balancing the state's reasons to end--or "interrupt," as cryonics enthusiasts would have it--his own life was not necessary. The trial court dismissed the complaint for failure to state a cause of action, and Donaldson appealed to the California Court of Appeals. Because the cryonic process would necessarily involve physician-assisted death, or the aiding, advising, or encouraging of another to commit suicide, the appellate court affirmed the ruling of the trial court, holding that Donaldson did not have a constitutional right to assisted death. Additionally, in light of Donaldson's First Amendment challenge to the statute, the court upheld the criminal statute prohibiting the aiding, advising, or encouraging of another to commit suicide. This Note briefly discusses the process of cryonic suspension and explores the holding of Donaldson in light of the underlying rationale of the California right-to-die cases. Considering the contradictory state and individual interests balanced in the right-to-die cases, this Note concludes that, under a similar balancing test, premortem cryonic suspension could be permitted under certain circumstances; however, the right to premortem cryonic suspension may be more effectively recognized through legislation.

[The successful prolongation of a twin preterm pregnancy complicated by a dead fetus and disseminated intravascular coagulation]. / Prolongación exitosa de gestación gemelar de pretérmino complicada con un feto muerto y coagulación intravascular diseminada.

A clinical case of twin pregnancy with one in utero death fetus, at 24 weeks of gestation is presented, accompanied by disseminated intravascular coagulation. The successful treatment with heparin its described.