Isoforms of 11beta-hydroxysteroid dehydrogenase in human granulosa-lutein cells.

To date, two isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD) have been characterized: a low affinity, NADP+-dependent isoform (11betaHSD1) and a high affinity, NAD+-dependent isoform which metabolizes dexamethasone and is inhibited by cortisone (11betaHSD2). Having previously reported a relationship between ovarian 11betaHSD activities and conception in women undergoing in vitro fertilization (IVF-ET), the objective of the present study was to identify which isoforms of 11betaHSD metabolize glucocorticoids in cultures of human granulosa-lutein cells. In both intact cells and cell homogenates, two distinct 11betaHSD activities were identified with differing affinities for cortisol (Km = 490 nM and 2.6 microM). Even at low concentrations, cortisol oxidation was preferentially supported by NADP+ and was independent of NAD+. Although inhibited by the hemisuccinate ester of glycyrrhetinic acid, carbenoxolone, the predominant 11betaHSD activity in intact cells was resistant to end-product inhibition. Intact cells were also able to reduce [3H]cortisone (Km = 190 nM) but did not metabolize [3H]dexamethasone. 11BetaHSD1 mRNA was expressed in 23 of 28 cell cultures whereas 11betaHSD2 mRNA was not expressed in any of the 22 independent cultures studied by reverse transcriptase-polymerase chain reaction (RT-PCR). We conclude that human granulosa-lutein cells express both type 11betaHSD and a novel isoform of this enzyme. While the low affinity 11beta-dehydrogenase and 11-ketosteroid reductase activities exhibit properties consistent with 11betaHSD1, the high affinity 11beta-dehydrogenase differs from 11betaHSD2 in that it is NADP+-dependent, does not metabolize dexamethasone and is resistant to end-product inhibition.

Folliculocentesis: a novel research technique to investigate the intrafollicular endocrine microenvironment.

During development of the dominant follicle, the avascular granulosa cells and oocyte are exposed to the follicular fluid endocrine microenvironment. An alteration in the endocrine characteristics of follicular fluid affects follicular steroidogenesis, oocyte maturation, ovulation and subsequent corpus luteum function. In-vitro studies on pooled follicular fluid from ovarian specimens lacked temporal precision between menstrual and follicular endocrine events. We have established a new technique, termed folliculocentesis (FC), to sample follicular fluid from the dominant ovarian follicle without compromising its growth or function during the mid- to late follicular phase. A total of 38 subjects with regular ovulatory cycles each underwent two identical cycles of hormone and follicle growth monitoring: one cycle served as the control, and FC was performed during the second cycle. During all cycles, plasma luteinizing hormone (LH), oestradiol and ultrasound monitoring of follicle growth were commenced on day 7 and continued until after ovulation. During FC cycles, 200 microliters of follicular fluid were aspirated from the dominant follicle using transvaginal ultrasound guidance when the follicle diameter reached > or = 10 mm. Six subjects were excluded from the study because of incomplete or invalid endocrine data. In all, 32 subjects completed both the FC and control cycles. The follicle growth pattern, maximum follicle diameter, plasma oestradiol, oestradiol peak, plasma LH, LH surge and follicular phase length were similar during FC and control cycles. A total of 50 valid follicular fluid samples were obtained when the dominant follicle was sampled once, twice or three times during the same cycle and from the same follicle in 15, 16 and one subjects respectively. The follicular fluid samples contained steroid concentrations consistent with those of the mid- to late follicular phase. We conclude that the FC procedure is safe, easy to perform and does not affect follicle growth or hormone dynamics. Analysis of the follicular fluid samples is expected to provide us with valuable in-vivo information about ovarian endocrinology.

Is analgesia required for transvaginal single-follicle aspiration in in vitro fertilization? A double-blind study.

High oocyte retrieval rates using transvaginal follicular aspiration for single follicles and improved laboratory techniques have engendered renewed interest in natural-cycle in vitro fertilization (IVF). To assess analgesia requirements during single-follicle aspiration, a double-blind study was set up in 30 patients comparing intraprocedure intravenous fentanyl with normal saline as a placebo. Analysis of pain perception using visual analogue scoring showed a similar pain tolerance in both groups for the procedures of vaginal ultrasound scanning, needle insertion, and follicular aspiration. A correlation between each patient's tolerance of common pain-producing experiences with that for the procedure was not well defined (r = 0.5). We conclude that one of the benefits of natural-cycle IVF using transvaginal single-follicle aspiration is that it can be performed without analgesia.