Effect of long-term exposure at suprazero temperatures on activity and viability of human ovarian cortex.

The experiment was designed to determine the optimal time and temperature for long-distance transport. Prolonged suprazero temperature exposure of ovarian tissue for 26 hours has no negative influence on follicle quality.

Cryobanking of human ovarian tissue for anti-cancer treatment: comparison of vitrification and conventional freezing.

The first case of cryopreservation of human ovarian tissue with good survival of follicles after warming was described in 1996. Childbirth after cryopreservation of ovarian tissue is now a reality. Cryopreservation of ovarian tissue can be performed using one of two methods: conventional ("slow") freezing and cryopreservation by direct plunging into liquid nitrogen (so called vitrification or "rapid" freezing). Comparative investigations of vitrification and conventional freezing performed on mammalian ovarian tissue are limited, and authors present different conclusions. The higher effectiveness of vitrification in comparison with conventional freezing for human oocytes and embryos was shown, whereas data on human ovarian tissue are limited. The aim of different studies was to compare the safety and effectiveness of conventional freezing and vitrification of human ovarian tissue. Below we shortly summarize the results of some investigations with different conclusions. The discussion on the post-warming quality of follicles as well as on the problems of microbial contamination of cells in liquid nitrogen at vitrification is presented. In our opinion, for cryopreservation of human ovarian tissue, conventional freezing is more promising than vitrification.

Effects of testosterone on hormonal content and calcium-dependent basal secretion in female rat pituitary cells.

In vivo and in vitro effects of elevated androgens on agonist-induced gonadotropin secretion have been addressed previously. Here we investigated the effects of testosterone on hormonal content and basal (in the absence of agonists) hormone release in pituitary lactotrophs, somatotrophs and gonadotrophs from female rats. Furthermore we tested the hypothesis that testosterone action is dependent on the pattern of spontaneous and Bay K 8644 (a L-type calcium channel agonist) -induced calcium signalling. Mixed anterior pituitary cells were cultured in steroid containing or depleted media, and testosterone (1pM to 10nM) was added for 48h. Cells were studied for their spontaneous and Bay K 8644-induced calcium signalling pattern and total hormone levels (release and hormonal content). In lactotrophs, somatotrophs and gonadotrophs testosterone did not affect the pattern of spontaneous calcium signalling. Bay K 8644-induced calcium signalling and hormone release were not affected by testosterone. In both steroid-depleted and -containing medium, testosterone inhibited prolactin (PRL), luteinizing hormone (LH) and growth hormone (GH) cellular content and release in a dose-dependent manner, with IC(50)s in a sub-nanomolar concentration range. These results indicate that testosterone inhibits basal hormone release from lactotrophs, somatotrophs and gonadotrophs without affecting intracellular calcium signalling. This action of testosterone is not dependent on the presence of other steroid hormones.

Actions of gonadotropin-releasing hormone analogues in pituitary gonadotrophs and their modulation by ovarian steroids.

Recently, GnRH antagonists (GnRHant) like cetrorelix and ganirelix have been introduced in protocols of controlled ovarian hyperstimulation for assisted reproductive techniques to prevent premature luteinizing hormone (LH) surges. Here we tested, whether the actions of cetrorelix and the GnRH agonist (GnRHag) triptorelin in gonadotrophs are dependent on the steroid milieu. Furthermore, we characterized the actions of cetrorelix and triptorelin on LH secretion and the total LH pool. Female rat pituitary cells were treated either with 0.1 nM triptorelin for 1, 2, 4 and 6 days or for 1, 3, 5 and 6 h or with 1, 10 or 100 nM cetrorelix for 1, 2, 3 and 5 h or for 10 min. Cells were stimulated for 3h with different concentrations of GnRH (10 pM-1 microM). For analysis of the total LH pool, which is composed of stored and released LH, cells were lysed with 0.1% Triton X-100 at -80 degrees C overnight. To test, whether the steroid milieu affects the actions of cetrorelix and triptorelin, cells were incubated for 52 h with 1 nM estradiol (E) alone or with combinations of 100 nM progesterone (P) for 4 or 52 h, respectively. Cells were then treated with 0.1 nM triptorelin for 9 h or 1 nM cetrorelix for 3 h and stimulated for 3 h with different concentrations of GnRH (10 pM-1 microM). The suppressive effect of triptorelin on LH secretion was fully accomplished after 3 h of treatment, for cetrorelix only 10 min were sufficient. The concentration of cetrorelix must be at least equimolar to GnRH to block LH secretion. Cetrorelix shifted the EC50s of the GnRH dose-response curve to the right. Triptorelin suppressed total LH significantly (from 137 to 36 ng/ml) after 1 h in a time-dependent manner. In contrast, only high concentrations of cetrorelix increased total LH. In steroid treated cells the suppressive effects of triptorelin were more distinct. One nanomolar cetrorelix suppressed GnRH-stimulated LH secretion of cells not treated with steroids from 10.1 to 3.5 ng/ml. In cells, additionally treated with estradiol alone or estradiol and short-term progesterone, LH levels were higher (from 3.5 to 5.4 or 4.5 ng/ml, respectively). In cells co-treated with estradiol and progesterone for 52 h LH secretion was only suppressed from 10.1 to 9.5 ng/ml. Steroid treatments diminished the suppressive effect of cetrorelix on LH secretion. In conclusion, the depletion of the total LH pool contributes to the desensitizing effect of triptorelin. The actions of cetrorelix and triptorelin are dependent on the steroid milieu.

Treatment strategies in PCOS patients.

Polycystic ovary syndrome (PCOS), with a prevalence of up to 7%, is the most common endocrinopathy in women of reproductive age. It is a complex metabolic-endocrine disorder with severe long-term health consequences, such as a higher risk of type 2 diabetes and cardiovascular diseases. According to prospective studies, women with PCOS have abnormal glucose tolerance and diabetes mellitus in 31.0-35.0% and 7.5-10.0% respectively. This risk is 2-3 times higher than normal. Insulin resistance plays a key role in the pathophysiology of this syndrome, and this makes the use of oral antidiabetic drugs most compelling. The majority of studies have shown amelioration of typical symptoms such as hyperandrogenism and cycle irregularities following the use of oral anti-diabetics, and ovulation and pregnancy rates increased. Furthermore, these drugs might be cardioprotective by improving insulin sensitivity and reducing the risk for type 2 diabetes. The best-investigated drug is metformin. Metformin is not approved for PCOS treatment in Germany and is a class B drug in pregnancy. In sterile PCOS patients, clomiphene citrate is still the first choice. The combination of clomiphene with metformin and lifestyle changes such as weight reduction and exercise might be superior to clomiphene alone. This article covers the use of different oral anti-diabetic drugs in the treatment of PCOS, and their influence on fertility and long-term health.

Gonadotropin-releasing hormone antagonists: pharmacology and clinical use in women.

Gonadotropin-releasing hormone (GnRH) antagonists are now widely used in protocols of patients with controlled ovarian hyperstimulation to treat infertility. By competitively binding to the pituitary GnRH receptor, they lead to a rapid suppression of gonadotropins and consecutively sex hormones. In the past, GnRH agonists have been exclusively used for these patients, with the disadvantage of an initial rise of gonadotropins--the flare-up effect. Several trials comparing the agonistic and antagonistic analogs of GnRH found no significant differences in oocyte quality, fertilization and pregnancy rates. Slightly lower implantation and pregnancy rates, and estradiol levels, in patients treated with GnRH antagonists has raised concern about eventual extrapituitary adverse effects. However, no convincing evidence has yet been found for any detrimental ovarian effects of GnRH antagonists. The lower rate of ovarian hyperstimulation syndrome, a potentially severe disadvantage of infertility treatment, is a positive feature of GnRH antagonists. The key point is that GnRH antagonists have been proven to be as effective and safe as GnRH agonists. This broadens the spectrum of indications for GnRH antagonists to sex hormone-dependent disorders like endometriosis, uterine fibroids, and gynecological cancers such as breast and ovarian cancer.