Elective cryopreservation of all pronuclear oocytes after GnRH agonist triggering of final oocyte maturation in patients at risk of developing OHSS: a prospective, observational proof-of-concept study.

BACKGROUND: A bolus dose of GnRH agonist can substitute for hCG as a trigger for the resumption of meiosis in ovarian stimulation with GnRH antagonists, which has been suggested to reduce the risk of ovarian hyperstimulation syndrome (OHSS). As the efficacy of this measure in fresh embryo transfer (ET) cycles is unclear, we evaluated a new clinical concept of GnRH-agonist triggering. METHODS: In this prospective, observational proof-of-concept study, 20 patients considered at increased risk of developing OHSS (> or = 20 follicles > or = 10 mm or estradiol > or = 4000 pg/ml, or a history of cycle cancellation due to OHSS risk or the development of severe OHSS in a previous cycle) after ovarian stimulation and concomitant GnRH-antagonist administration had final oocyte maturation triggered with 0.2 mg triptorelin s.c. All two pronucleate (2 PN) oocytes were cryopreserved by vitrification, and frozen-thawed ETs (FT-ETs) were performed in an artificial cycle. Main outcome measures were the cumulative ongoing pregnancy rate per patient and the ongoing pregnancy rate per first ET. Secondary outcomes included the incidence of moderate-to-severe OHSS. RESULTS: Of the 20 patients triggered with GnRH agonist, 19 patients underwent 24 FT-ETs in the observational period. The cumulative ongoing pregnancy rate was 36.8% (95% confidence interval: 19.1-59.0%). The ongoing pregnancy rate per first FT-ET was 31.6% (15.4-54.0%). No cases of moderate or severe OHSS were observed. CONCLUSIONS: The present study is the proof of the concept that GnRH-agonist triggering of final oocyte maturation in combination with elective cryopreservation of 2 PN oocytes offers OHSS risk patients a good chance of pregnancy achievement, while reducing the risk of moderate and severe OHSS.

GnRH agonists and antagonists in assisted reproduction: pregnancy rate.

Although gonadotrophin-releasing hormone (GnRH) antagonists offer many advantages when used in ovarian stimulation, their popularity is lower than expected. GnRH protocols are suspected to yield lower pregnancy rates compared with the long agonist protocol. In the current study, subgroup analyses from the German IVF registry (DIR) were performed to evaluate the hypothesis that GnRH antagonists are often used as second-line medication in patients with difficult medical conditions, and thus pregnancy rates may be biased. Consequently, pregnancy rates in the first six stimulation cycles (low rank cycles) were more favourable in the group treated according to the long protocol, while in the patient group requiring more stimulation cycles (high rank cycles; 7, 9 and 10), numerically higher pregnancy rates were achieved with GnRH antagonist protocols. On the other hand, in a patient collective with equal demographic and clinical features (<35 years, tubal infertility), the long and the GnRH antagonist protocols resulted in similar pregnancy rates, supporting the hypothesis that both stimulation protocols lead to equal results.

Comet assay of cumulus cells and spermatozoa DNA status, and the relationship to oocyte fertilization and embryo quality following ICSI.

It has been postulated that apoptosis may affect cumulus cell and sperm DNA integrity, and therefore influence the outcome of assisted reproductive techniques. This study investigates apoptotic levels in both cumulus cells and spermatozoa, and their relationship with fertilization and embryo quality after intracytoplasmic sperm injection (ICSI). The neutral comet assay was performed on cumulus cells and semen samples from 55 couples with male factor infertility undergoing ICSI treatment. Cells were fixed in agarose on comet assay slides, lysed in a neutral buffer and submitted to electrophoresis. The cells were stained with SYBR green fluorescent dye, which binds to double-stranded DNA and upon excitation emits light. Analysis showed that there was no correlation between apoptosis levels and the outcome of ICSI (fertilization and embryo quality).

A lower ongoing pregnancy rate can be expected when GnRH agonist is used for triggering final oocyte maturation instead of HCG in patients undergoing IVF with GnRH antagonists.

BACKGROUND: Eliciting an endogenous LH surge by GnRH-agonist for the induction of final oocyte maturation may be more physiological compared with the administration of HCG. However, the efficacy of this intervention in patients treated for IVF with GnRH antagonists remains to be assessed. METHODS: 106 patients were randomized to receive either 10 000 IU urinary HCG or 0.2 mg Triptorelin for triggering final oocyte maturation. Ovarian stimulation for IVF was performed with a fixed dose of 200 IU recombinant FSH and GnRH antagonist was started on stimulation day 6. Luteal phase was supported with micronized vaginal progesterone and oral estradiol. The study was monitored continuously for safety and stopping rules were established. RESULTS: No significant differences were present in the number of cumulus-oocyte complexes retrieved, in the proportion of metaphase II oocytes, in fertilization rates or in the number and quality of the embryos transferred between the two groups. However, a significantly lower probability of ongoing pregnancy in the GnRH agonist arm prompted discontinuation of the trial, according to the stopping rules established (odds ratio 0.11; 95% confidence interval 0.02-0.52). CONCLUSIONS: Lower probability of ongoing pregnancy can be expected when GnRH agonist is used for triggering final oocyte maturation instead of HCG in patients undergoing ovarian stimulation for IVF with GnRH antagonists.

[Treatment of breast cancer patients with brain metastases]. / Behandlung von zerebralen Metastasen beim Mammakarzinom.

Treatment of patients with brain metastases is based on an interdisciplinary approach. It is essential to perform a differenciated indication for procedures with consideration of all therapeutical options like modalities of radiotherapy, surgery and oncology. In the case of multiple brain metastases the whole brain radiotherapy is the standard of treatment, while in case of a single or solitary brain metastasis surgical procedures followed by radiotherapy should be preferred.

Recombinant luteinizing hormone supplementation to recombinant follicle-stimulating hormone induced ovarian hyperstimulation in the GnRH-antagonist multiple-dose protocol.

BACKGROUND: Suppression of endogenous LH production by mid-follicular phase GnRH-antagonist administration in controlled ovarian hyperstimulation protocol using recombinant (rec) FSH preparations void of LH activity may potentially affect ovarian response and the outcome of IVF treatment. The present study prospectively assessed the effect of using a combination of recFSH and recLH on ovarian stimulation parameters and treatment outcome in a fixed GnRH-antagonist multiple dose protocol. METHODS: 127 infertile patients with an indication for IVF or ICSI were recruited and randomized (using sealed envelopes) to receive a starting dose of either 150 IU recFSH (follitropin alpha) or 150 IU recFSH plus 75 IU recLH (lutropin alpha) for ovarian hyperstimulation. GnRH-antagonist (Cetrorelix) 0.25 mg was administered daily from stimulation day 6 onwards up to and including the day of the administration of recombinant HCG (chorion gonadotropin alpha). Gonadotropin dose adjustments were allowed from stimulation day 6 onwards, HCG was administered as soon as three follicles > or =18 mm were present. The primary outcome parameter was treatment duration until administration of HCG. RESULTS: Exogenous LH did not shorten the time necessary to reach ovulation induction criteria. Serum estradiol (E(2)) and LH levels were significantly higher on the day of HCG administration in the recLH-supplemented group (1924.7 +/- 1256.4 vs 1488.3 +/- 824.0 pg/ml, P < 0.03), and 2.1 +/- 1.4 vs 1.4 +/- 1.5 IU/l, P < 0.01, respectively). CONCLUSIONS: Except for higher E(2) and LH levels on the day of HCG administration, no positive trend in favour of additional LH was found as defined by treatment outcome parameters.

Five years' clinical use of GnRH antagonists: evaluation of safety and allergic potential.

Three antagonists of gonadotrophin-releasing hormone are currently clinically available. Cetrorelix (Cetrotide) and ganirelix (Orgalutran/Antagon) have been safely used in assisted reproduction since 1999 and 2000 respectively. The structurally similar abarelix (Plenaxis) has been approved for the therapy of advanced androgen sensitive prostate cancer. However, due to the occurrence of allergic reactions, its use is restricted to only a subgroup of patients. These allergic side effects may not be due to abarelix, as the drug itself does not have a strong histamine liberating potential in vitro, but could be attributed to carboxymethylcellulose (CMC), which only Plenaxis, but not Cetrotide or Orgalutran/Antagon contain. CMC is used to obtain the sustained-release characteristics of Plenaxis. Since 1973, numerous case reports and studies have been published regarding allergic reactions and specific immunoglobulin E antibodies against CMC. Thus, the allergic side effects of Plenaxis could be rather due to CMC than to abarelix.

FSH time-concentration profiles before and after administration of 0.25 mg cetrorelix in the gnRH-antagonist multiple-dose protocol for ovarian hyperstimulation.

PURPOSE: To determine FSH concentration behavior before and after cetrorelix 0.25 mg administration in the GnRH-antagonist multiple-dose protocol on day 6 of ovarian stimulation with 150-300 IU daily recombinant FSH. METHODS: Blood samples for FSH measurements were drawn from seven women every 15 min from 8 h prior to the first cetrorelix administration in the GnRH-antagonist multiple-dose protocol until 15-32 h thereafter. RESULTS: No significant change of FSH concentration was observed. CONCLUSIONS: This observation indicates that no rationale exists of increasing the daily FSH dosage concomitantly to the GnRH-antagonist administration to compensate for a drop of endogenous FSH.

Characterization of calcium-mobilizing, purinergic P2Y(2) receptors in human ovarian cancer cells.

In human ovarian EFO-21 and EFO-27 carcinoma cells, extracellular ATP induced a concentration-dependent rise in intracellular calcium concentration ([Ca(2+)](i)), suggesting the expression of a purinoreceptor. ATP and UTP were equipotent in generating [Ca(2+)](i) signals, followed by ATP-gamma-S and ADP, whereas beta, gamma-ATP, 2 methyl 1 thio-ATP, 3'-o-(4-benzoyl) benzoyl-ATP, AMP, and adenosine were ineffective. This pharmacological profile suggested the presence of the P2Y(2) subtype in both cell types, and this was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis using P2Y(2) primers. ATP-induced [Ca(2+)](i) signals were composed of two phases: an early and extracellular calcium-independent phase, followed by a sustained plateau phase that was dependent on capacitative calcium influx. In addition to the rise in the [Ca(2+)](i), a time- and concentration-dependent increase in phosphatidylethanol accumulation was observed in ATP-stimulated cells, indicating an increase in phospholipase D activity. RT-PCR analysis identified the expression of a transcript for the phospholipase D-1 subtype of this enzyme. Activation of these receptors by a slowly degradable analogue, ATP-gamma-S, attenuated basal and fetal calf serum-induced cell proliferation in a time- and concentration-dependent manner. These results indicate that ATP may act as an extracellular messenger in controlling the ovarian epithelial cell cycle through P2Y(2) receptors.

Expression and responsiveness of P2Y2 receptors in human endometrial cancer cell lines.

In single endometrial carcinoma HEC-1A and Ishikawa cells, ATP induced a rapid and extracellular Ca2+-independent rise in cytosolic Ca2+ concentration ([Ca2+]i) in a dose-dependent manner, with an ED50 of about 10 microM. The spike phase was followed by a sustained plateau phase that was dependent on Ca2+ influx through voltage-insensitive Ca2+ channels, whose gating was controlled by a capacitative Ca2+ entry mechanism. ADP was less potent in raising the cystolic Ca2+ concentration, and AMP and adenosine were ineffective. The order of agonist potency for this receptor was ATP = UTP > ATP-gamma-S >> ADP. Several other agonists, including beta,gamma-methylene-ATP, 2-MeS-ATP, and BzATP were ineffective. This ligand-selective profile indicates the expression of the P2Y2R subtype in endometrial cells. Accordingly, reverse transcription-PCR using P2Y2 primers amplified the expected transcript from both cell lines. The coupling of these receptors to phospholipase C was confirmed by the ability of ATP to increase inositol 1,4,5-trisphosphate and diacylglycerol productions. These receptors are also coupled to the phospholipase D-1 pathway, leading to accumulation of phosphatidic acid. Activation of P2Y2 receptors by a slowly degradable ATP analog, ATP-gamma-S, was associated with a significant suppression of cell proliferation without affecting the cellular apoptosis. These results indicate that P2Y2 receptors may participate in control of the cell cycle of endometrial carcinoma cells.

Paracrine changes in the peritoneal environment of women with endometriosis.

During the past decade, macrophage-derived substances such as prostanoids, cytokines, growth factors and angiogenic factors have been detected in the peritoneal fluid of women with endometriosis. In particular, growth-promoting and angiogenic factors are considered to be substantially involved in the pathogenesis of endometriosis. In this study, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta) and intercellular adhesion molecule 1 (ICAM-1), substances recently detected in the peritoneal fluid of women with endometriosis, were assessed with regard to their concentrations in different stages of endometriosis and changes of the peritoneal paracrine activity after medical treatment with a gonadotrophin releasing hormone agonist (GnRHa). Peritoneal fluid was obtained from patients with endometriosis during laparoscopy before and after a 4-month treatment with a GnRHa. VEGF, TGF-beta and ICAM-1 could be detected in all women presenting with various stages of active endometriosis. After GnRHa therapy, all patients showed significant decreases in mean concentrations of VEGF (194+/-77 pg/ml), TGF-beta (902+/-273 pg/ml) and ICAM-1 (157+/-52 ng/ml). Patients with stage III and IV endometriosis (according to the rAFS score) had much higher concentrations of VEGF and TGF-beta before treatment compared with those patients with mild endometriosis (rAFS stages I and II). The most striking decrease in concentration was for TGF-beta, from 902 pg/ml before to 273 pg/ml after therapy. These results indicate an important role for paracrine activity in the establishment and maintenance of endometriosis. Indeed, treatment with a GnRHa may reduce paracrine activity in the peritoneal cavity via hypo-oestrogenism and provide proof of successful therapy.