Resveratrol and desferoxamine protect human OxLDL-treated granulosa cell subtypes from degeneration.

CONTEXT: Obese women suffer from anovulation and infertility, which are driven by oxidative stress caused by increased levels of lipid peroxides and circulating oxidized low-density lipoprotein (oxLDL). OxLDL binds to lectin-like oxLDL receptor 1 (LOX-1), cluster of differentiation 36 (CD36), and toll-like receptor 4 (TLR4) and causes cell death in human granulosa cells (GCs). OBJECTIVE: Our objective was to reveal whether treatment with antioxidants resveratrol (RES) and/or desferoxamine (DFO) protect GCs from oxLDL-induced damage. DESIGN AND SETTING: This basic research study was performed at the Institute of Anatomy and the Clinic of Reproductive Medicine. PATIENTS: Patients were women undergoing in vitro fertilization therapy. MAIN OUTCOME MEASURES: GC cultures were treated with oxLDL alone or with RES or DFO under serum-free conditions for up to 36 hours. Dead cells were determined by propidium iodide uptake, cleaved caspase-3 expression, and electron microscopy. Mitosis was detected by Ki-67 immunostaining. LOX-1, TLR4, CD36, and heat-shock protein 60 were examined by Western blot. Measurement of oxidative stress markers (8-iso-prostaglandin F2α, advanced glycation end products, and protein carbonyl content) was conducted with ELISA kits. RESULTS: Different subtypes of human GCs exposed to RES or DFO were protected as evidenced by the lack of cell death, enhanced mitosis, induction of protective autophagy, reduction of oxidative stress markers, and reduced expression of LOX-1, TLR4, CD36, and heat-shock protein 60. Importantly, RES could restore steroid biosynthesis in cytokeratin-positive GCs, which exhibited significant induction of steroidogenic acute regulatory protein. CONCLUSIONS: RES and DFO exert a protective effect on human GCs. Thus, RES and DFO may help improve the treatment of obese women or polycystic ovarian syndrome patients undergoing in vitro fertilization therapy.

Granulosa cell subtypes vary in response to oxidized low-density lipoprotein as regards specific lipoprotein receptors and antioxidant enzyme activity.

CONTEXT: The oxidized low-density lipoprotein (oxLDL) and its lectin-like oxLDL receptor-1 (LOX-1) are found in the follicular fluid and in granulosa cells. Lipoprotein receptors and antioxidant enzymes could differ in granulosa cell subtypes. OBJECTIVE: Our aim was to reveal cell-specific responses under oxLDL treatment. DESIGN AND SETTING: We conducted basic research at the Institute of Anatomy and the Clinic of Reproductive Medicine. PATIENTS: Women undergoing in vitro fertilization therapy participated in the study. MAIN OUTCOME MEASURES: Cultures of cytokeratin-positive/negative (CK(+)/CK(-)) granulosa cells and of cumulus cells were treated with 150 microg/ml oxLDL or native LDL under serum-free conditions for up to 36 h. Dead cells were determined by uptake of propidium iodide. LOX-1, toll-like receptor 4, and cluster of differentiation 36 (CD36) were examined in lysates by Western blots. The enzyme activities were determined in lysates and in supernatants. RESULTS: Under oxLDL treatment, predominantly CK(+) cells underwent nonapoptotic cell death. Receptors showed a cell-specific pattern of up-regulation: toll-like receptor 4 in CK(+) cells, LOX-1 in CK(-) cells, and CD36 in cumulus cells. An antioxidant ranking occurred: superoxide dismutase activity in CK(+) cells, total glutathione in CK(-) cells, and catalase activity in cumulus cells. The supernatants of oxLDL-treated CK(+) cell cultures contained more catalase activity than in controls, whereas a moderate increase was noted for glutathione peroxidase (GPx) in supernatants of CK(-) and cumulus cells. CONCLUSIONS: Catalase/GPx activity in the supernatants may be due to cell death or to secretion. Oxidative stress could be sensed by CK(+) cells and indicated by changes in catalase/GPx activity in the follicular fluid during ovarian disorders.

Effects of post-density gradient swim-up on apoptosis signalling in human spermatozoa.

The inclusion of apoptotic spermatozoa during assisted reproductive techniques (ART) may be one reason for suboptimal success rates. The aim of our study was to evaluate the potential of routine semen preparation to eliminate spermatozoa with activated apoptosis signalling. Semen samples from 20 infertility patients scheduled for ART procedures were investigated. Following density gradient centrifugation (DGC) and swim-up, aliquots were taken from each sample to analyse motility, Caspase-3 activation (CP3) and integrity of the mitochondrial membrane potential (MMP) using flow cytometry. Aliquots from the neat semen served as controls. Semen samples of patients contained 53.8 +/- 17.7% spermatozoa with disrupted MMP and 51.8 +/- 14.9% with active CP3. Preparation by DGC and swim-up resulted in improvement of progressive motility (+43.5%) and reduction of spermatozoa with disrupted MMP (-34.3%) and activated CP3 (-25.7%, P < 0.01). Minimal reduction of spermatozoa with disrupted MMP and active CP3 was 6.0% and 0.7%, maximum reduction was 65.5% (disrupted MMP) and 49.3% (CP3). Semen samples of subfertile patients contain high levels of spermatozoa with activated apoptosis signalling. Although there was a reduction in the majority of the samples, profound interindividual differences in the separation effect demand further development of innovative molecular-based separation methods to deplete apoptotic spermatozoa.

Eosinophils in the human corpus luteum: the role of RANTES and eotaxin in eosinophil attraction into periovulatory structures.

We evaluated the presence and number of eosinophils at varying stages in the human corpus luteum from 27 ovaries of women at reproductive age. Eosinophils preferentially accumulated in dilated microvessels of the thecal layer transforming into septa of the corpus luteum. The granulosa layer under luteinization, the thecal layer, and haemorrhages in the former antrum each contained low, moderate and high numbers of extravasated eosinophils respectively. Eosinophils decreased rapidly during the stages of secretion and regression. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) systems were used to investigate the expression and regulation of the eosinophil-attracting chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin in granulosa cells obtained from follicular aspirates from women undergoing IVF. Contaminating leukocytes were determined by CD18 mRNA quantification. Granulosa cells expressed RANTES (n = 3; 43 +/- 14 pg/ml, mean +/- SEM). 4ss-phorbol-12-myristate-13-acetate (PMA; 211 +/- 53) and tumour necrosis factor alpha (TNFalpha) (238 +/- 59), but not interleukin (IL)-1 up-regulated RANTES at significant levels. In general, higher basal and stimulated RANTES mRNA and protein were found in cultures with higher CD18 mRNA levels than in those with lower levels. We found only traces of eotaxin mRNA and no eotaxin secretion, even in stimulated granulosa cell cultures, independently of leukocyte levels. Taken together, this is the first study demonstrating the selective presence of eosinophils in human periovulatory structures. RANTES, but not eotaxin, may play an active process in the accumulation of these cells.

[Initial experiences with intra-tubal gamete transfer (GIFT)]. / Erste Erfahrungen mit dem intratubaren Gametenfransfer (GIFT).

A gamete intra-fallopian transfer was performed in 13 women. In all these patients the ovulation was stimulated according to our IVF programme. For the transfer procedure we used culture medium in eight cases. This medium was replaced by follicular fluid in five other cases. Altogether the GIFT resulted in seven pregnancies, two in the culture medium group and five in the follicular fluid group. The advantages of the follicular fluid as transfer medium are discussed.

[Detection of dipeptidyl peptidase IV in follicular fluid--initial report]. / Der Nachweis der Dipeptidyl-Peptidase IV in der Follikelflüssigkeit--ein erster Bericht.

Aspirates of ovarian follicles were obtained from patients subjected to gynaecologic surgeries. This is the first report of dipeptidyl-peptidase IV (DP IV) activity in human follicular fluid. This enzyme specifically cleaves regulatory important oligo- and polypeptides. Only from peptides with proline or alanine in position P1 of the N-terminal sequence a dipeptide is split off. DP IV activity was measured with glycyl-L-proline p-Nitroanilide as substrate. Levels in a range of 209.8 +/- 57.4 nmol l-1 s-1 were achieved.