The regulation of adrenocorticotrophic hormone receptor by corticotropin-releasing hormone in human fetal adrenal definitive/transitional zone cells.

As gestation progresses, human fetal adrenals (HFA) initiate the production of cortisol, which increases placental corticotropin-releasing hormone (CRH) biosynthesis. While adrenocorticotrophic hormone (ACTH) is important for the onset of cortisol production, the late gestational surge in cortisol production occurs despite falling ACTH levels in the fetal circulation. The authors determine if CRH directly regulates the expression of the ACTH receptor (ACTHR) in HFA definitive/transitional zone (DZ/TZ) cells. DZ/TZ cells isolated from midgestation HFA were cultured before treatment with 0.01 nM to 100 nM CRH or ACTH. Cortisol was measured by radioimmunoassay. Real-time reverse-transcriptase polymerase chain reaction was used to measure ACTHR mRNA. Whole-cell ACTH binding studies were performed using I(125) (Tyr-23) ACTH. CRH produced a dose-dependent rise in cortisol production and caused a time-dependent increase in ACTHR mRNA levels between 12 and 24 hours. As little as 0.1 nM CRH induced ACTHR transcript by 12-fold at 24 hours. Together with ACTH 0.01 nM, 0.03 or 0.1 nM CRH increased ACTHR expression more than ACTH alone. Binding assays demonstrated a 3.5-fold increase in ACTHR protein at 48 hours with combined CRH and ACTH treatment. Physiologic levels of CRH seen in the late-gestation fetus stimulate DZ/TZ ACTHR expression. Since placental CRH production increases strikingly near the end of gestation, the authors suggest that CRH-induced ACTH receptor expression may increase TZ responsiveness to circulating ACTH and contribute to the late gestational rise in cortisol secretion by the HFA, participating in an endocrine cascade that is involved in fetal organ maturation and potentially in the timing of human parturition.

Late stages of embryo progression are a much better predictor of clinical pregnancy than early cleavage in intracytoplasmic sperm injection and in vitro fertilization cycles with blastocyst-stage transfer.

OBJECTIVE: To define and validate metrics of embryo progression and morphology during extended embryo culture and to compare the effects of early cleavage (EC) vs. blastulation stages on clinical pregnancy. DESIGN: Retrospective observational study. SETTING: University-affiliated assisted reproduction center. PATIENT(S): One thousand two hundred ninety-two intracytoplasmic sperm injection and 842 IVF blastocyst-transfer cycles. INTERVENTION(S): The embryo progression index (EPI) was calculated as the area under the curve of total cell number (TCN) over time, by using observed TCN for cleavage-stage embryos and estimated blastocyst TCN according to morphology. The EPI from days 1-3 measured early cleavage, and blastulation was assessed by EPI over extended embryo culture. Blastocyst morphology was converted into numerical blastocyst quality scores (BQSs). Receiver operating characteristic curve analysis was used to evaluate predictors for clinical pregnancy. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): Per-cycle mean EPI and mean BQS for all embryos developing into blastocysts, as well as mean BQS of the transferred embryos, were significant predictors of clinical pregnancy in intracytoplasmic sperm injection and IVF cycles. Mean EPI for days 1-3 did not predict outcome. CONCLUSION(S): Early cleavage is a putative marker of embryo quality. Late-stage embryo development is more sensitive and specific in predicting clinical pregnancy than is early cleavage, supporting the use of extended embryo culture for embryo selection. The embryo progression index and BQS may also be used for this purpose.

Precycle administration of GnRH antagonist and microdose HCG decreases clinical pregnancy rates without affecting embryo quality and blastulation.

The outcome of a novel protocol utilizing precycle gonadotrophin-releasing hormone (GnRH) antagonist administration and LH activity support with microdose recombinant human chorionic gonadotrophin (HCG) was compared to GnRH agonist long protocol used in patients undergoing their first ICSI (n=707) or IVF (n=571) cycles, which had resulted in one or two blastocyst transfers. In GnRH antagonist cycles, cetrorelix acetate (3 mg) was administered s.c. 4 days before FSH stimulation and a repeat dose was given when the lead follicular diameter was 13-14 mm. LH support was provided by recombinant HCG (2.5 microg). Embryo progression and blastulation were evaluated using embryo progression indices and blastocyst quality scores. The tested protocol demonstrated reduced implantation and clinical pregnancy rates as compared with GnRH agonist long protocol, although the embryo progression and blastulation parameters and blastocyst quality were comparable among the groups. Logistic regression models further supported the significant negative impact of GnRH antagonist/microdose HCG protocol on clinical pregnancy rates in both ICSI and IVF patients. Assisted reproduction cycles with fresh blastocyst transfers utilizing precycle GnRH antagonist administration and microdose HCG support resulted in lower implantation and clinical pregnancy rates as compared with GnRH agonist cycles, although the embryo progression and blastulation parameters were comparable.

Corticotropin-releasing hormone directly stimulates cortisol and the cortisol biosynthetic pathway in human fetal adrenal cells.

Near term the human fetal adrenals (HFAs) initiate production of cortisol, which promotes organ maturation and acts to increase placental CRH biosynthesis. The objective of the present study was to determine whether CRH directly stimulates both cortisol production and expression of the steroidogenic enzymes in HFA-definitive zone cells. CRH stimulated the production of cortisol in a time- and dose-dependent manner, with an effective concentration of as low as 0.01 nm. In real-time RT-PCR experiments, CRH treatment increased the mRNA levels of steroidogenic acute regulatory protein and each of the enzymes needed to produce cortisol. CRH induced 3beta-hydroxysteroid dehydrogenase type II (HSD3B2) by 34-fold, 21-hydroxylase (CYP21) by 55-fold, and 11beta-hydroxylase by 41-fold. Induction of steroidogenic acute regulatory protein, cholesterol side chain cleavage (CYP11A), and 17alpha-hydroxylase (CYP17) mRNA by CRH was 6-, 4-, and 6-fold, respectively. We also demonstrated that submaximal concentrations of CRH (30 pm) and ACTH (30 pm) that are seen in fetal circulation were additive on cortisol biosynthesis and 3beta-hydroxysteroid dehydrogenase type II mRNA induction. We suggest that CRH may play an important role in the late gestational rise in cortisol secretion from the HFAs, which may serve to augment placental CRH production and therefore participate in the endocrine cascade that is involved in fetal organ maturation and potentially in the timing of human parturition.

Fetal and maternal adrenals in human pregnancy.

Human pregnancy is marked by alterations in several endocrine systems--perhaps most notably, the striking increase in steroid hormone production by the adrenals of the fetus and mother. Morphologically and physiologically, the human fetal adrenal glands are remarkable organs. In proportion to the adult organs, the adrenal cortex is the largest organ of the fetus. At term, they produce more steroid and weigh the same as adrenal glands of the adult. Much of the steroid that is released by the fetal and maternal adrenals during pregnancy is the sulfated form of dehydroepiandrosterone (DHEA-S), which is used by the placenta to produce estrogens. Herein, we discuss the physiologic and pathophysiologic hormonal changes of the fetal and maternal adrenals during the course of pregnancy.

The human fetal adrenal: making adrenal androgens for placental estrogens.

During most of gestation, the fetal adrenal gland is almost solely dedicated to the production of dehydroepiandrosterone sulfate (DHEA-S). This specialized ability of the fetal adrenal is unique to primates and occurs because of a specialized fetal zone that composes the bulk of the fetal adrenal gland. Morphologically and physiologically, the human fetal adrenal (HFA) glands are remarkable organs. The glands at term are almost the size of the fetal kidney due in large part to the presence of the fetal zone, which at term produces more steroid than is normally secreted by adrenal glands of the adult. Much of the steroid released by the fetal zone is DHEA-S, which is used by the placenta to produce estrogens. Herein, we review the features of the HFA gland, including its impressive ability to synthesize large amounts of adrenal androgens for use by the placenta to produce estrogens.

Sex differences in adrenal androgens.

The primate adrenal cortex secretes high levels of 19 carbon (C19) steroids including dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and androstenedione. These steroids exhibit weak androgenic activity but serve as precursors for estrogens and active androgens such as testosterone. Thus, they are commonly known as adrenal androgens. Age-related changes in adrenal androgen production are well-described in humans and other primates. This article discusses the evidence for sex differences in adrenal androgen production in humans and both nonhuman primate and nonprimate animal models, which present varying degrees of sexual dimorphism in adrenal structure and function. Possible mechanisms underlying these gender differences and their relevance to human adrenocortical physiology will be discussed. Although animal and human studies have provided insight into the regulation of adrenal androgen production, the basis of the observed sex differences remains poorly understood. The putative modulation of adrenal androgen production by sex steroids merits further research, as does the possibility of gender-specific differences in adrenocortical zonation.

Human myometrial adaptation to pregnancy: cDNA microarray gene expression profiling of myometrium from non-pregnant and pregnant women.

The human uterus undergoes profound physiological tissue remodelling during pregnancy. In the myometrium, altered gene expression must underlie these extensive molecular and structural changes. The purpose of this study was to compare expression profiles of pregnant and non-pregnant myometrium, in order to identify genes that participate in this process. mRNA from 14 non-pregnant and four pregnant human myometrial samples were analysed using a human UniGEM V microarray with 7075 cDNA elements. A total of 602 transcripts from the microarray were up-regulated >/=2.0-fold in pregnant myometrium, with 37 transcripts up-regulated >/=4.0-fold. In contrast, eight transcripts were down-regulated >/=2.0-fold in pregnancy. To ensure accurate representation of differential gene expression, Northern blot analyses using total RNA from 16 samples of non-pregnant and pregnant myometrium were used to examine mRNA levels for four of the genes that were differentially expressed by microarray analysis, namely plasminogen activator inhibitor type 1 (PAI-1), milk fat globule-EGF factor 8 protein (MFGE8), secreted frizzled-related protein 4 (sFRP4) and estrogen receptor alpha (ERalpha). On the microarray these transcripts were up-regulated 7.5-fold for PAI-1 and 4.9-fold for MFGE8 in pregnant myometrium, and down-regulated 3.7-fold for sFRP4 and 2.9-fold for ERalpha in pregnancy. Northern blot analyses confirmed these changes. Our findings suggest that microarray technology is a useful tool for examining global changes in gene expression that occur as the myometrium differentiates from non-pregnant to pregnant status. Defining these changes provides new insight into the structural and functional adaptations of human myometrium during pregnancy.

Profiling the steroidogenic pathway in human fetal and adult adrenals.

OBJECTIVE: Gene expression clearly underlies the marked structural and functional differences between the human fetal adrenal (HFA) and adult adrenal. We thus measured expression of steroidogenic enzymes and associated cofactors in these tissues. METHODS: Real-time reverse transcriptase polymerase chain reaction was used to quantify transcripts encoding steroidogenic enzymes and the cofactors steroidogenic acute regulatory protein (StAR), cytochrome b5 (CYb5), and P450 oxidoreductase (POR). RESULTS: Cholesterol side-chain cleavage mRNA levels were 1.9-fold higher in the HFA than in the adult adrenal. Compared with a nonsignificant difference in 17alpha-hydroxylase/17,20 lyase mRNA abundance, CYb5 and POR were expressed 2.3-fold and 2.0-fold higher, respectively, in the HFA. Dehydroepiandrosterone (DHEA) sulfotransferase transcript (SULT2A1) was present at 13-fold higher levels in the HFA than the adult. 3beta-Hydroxysteroid dehydrogenase type II (HSD3B2) mRNA was 127-fold higher in the adult adrenal. StAR, 21-hydroxylase, 11beta hydroxylase, and aldosterone synthase mRNA abundance did not differ significantly. CONCLUSION: In the HFA, increased mRNA for cholesterol side-chain cleavage reflects high cholesterol utilization for steroidogenesis. Both CYb5 and POR cofactors may up-regulate 17alpha-hydroxylase/17,20 lyase activity and thus DHEA sulfate production in the HFA. High levels of SULT2A1 mRNA reflect high DHEA sulfonation in the HFA and restricted expression in the adult. Lack of HSD3B2 in the HFA facilitates DHEA synthesis. The novel finding of high levels of 21-hydroxylase and 11beta hydroxylase transcripts in the midgestational HFA merits further investigation. Thus different patterns of steroidogenic enzyme and cofactor gene expression might account for some of the phenotypic differences between the fetal and adult adrenal.

Gene therapy and reproductive medicine.

OBJECTIVE: To review the literature on the principles of gene therapy and its potential application in reproductive medicine. DESIGN: Literature review. SETTING: Gene therapy involves transfer of genetic material to target cells using a delivery system, or vector. Attention has primarily focused on viral vectors. Significant problems remain to be overcome including low efficacy of gene transfer, the transient expression of some vectors, safety issues with modified adenoviruses and retroviruses, and ethical concerns. If these issues can be resolved, gene therapy will be applicable to an increasing spectrum of single and multiple gene disorders, as the Human Genome Project data are analyzed, and the genetic component of human disease becomes better understood. Gynecologic gene therapy has advanced to human clinical trials for ovarian carcinoma, and shows potential for the treatment of uterine leiomyomata. Obstetric applications of gene therapy, including fetal gene therapy, remain more distant goals. CONCLUSION(S): Concerns about the safety of human gene therapy research are being actively addressed, and remarkable progress in improving DNA transfer has been made. The first treatment success for a genetic disease (severe combined immunodeficiency disease) has been achieved, and ongoing research efforts will eventually yield clinical applications in many spheres of reproductive medicine.