Female fertility: clinical aspects

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controlled ovarian hyperstimulation for IVF/ICSI and were isolated by centri- fugation over percoll ..... Analysis of his semen showed a teratozoospermia. The.

Abstracts of the 21st Annual Meeting of the ESHRE, Copenhagen, Denmark, 19–22 June 2005 regulated by voltage-dependent Ca2+ channels (VDCC). The type of voltage-dependent Ca2+ channel on Leydig cells is not completely identified. The objective of this study is to identify the type of Ca2+ channel that may lead to the induction of steroidogenesis in hCG-stimulated Leydig cells. Materials and methods: Leydig cells were prepared from adult mice by a mechanical method. The purified Leydig cells were incubated in DMEM/F12 medium supplemented with 10% bovine serum at 31 C in 5% CO2 for 3 days. At this point, the culture medium was replaced with a fresh medium containing hCG (1 IU or 10 IU) and Mibefradil (0.01 mM/ml, 0.1 mM/ml, or 1 mM/ml), a putative T-type Ca+2 channel blocker, and further incubated for 30 min or 1 h. As a control, Leydig cells were incubated in a Ca2+ free medium supplemented with hCG for 30 min or 1 hr. After the treatment, the cells were collected and analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for the expression of steroidogenic acute regulatory protein (StAR) gene. The conditioned media were collected and assayed for the presence of testosterone by radioimmunoassay. Results: hCG significantly elevated the testosterone level in the culture media of Leydig cells, and this stimulatory effect of hCG was significantly blocked by a T type VDCC blocker, Mibefradil in a dose-dependent manner whereas no hCG-induced increase in testosterone productivity was observed in a Ca2+-free medium. The treatment of hCG also increased the StAR gene expression in Leydig cells dose-dependently. Therefore, the hCG-induced enhancement of steroidogenesis in Leydig cells appeared to be dependent on the presence of Ca2+ in the external medium and the entry of Ca2+ through the T-type VDCC in order to enhance the steroidogenesis. Conclusion: Our data showed the evidence that supports a close functional relationship between the hCG-induced steroidogenesis and the Ca2+ entry through the VDCC in mouse Leydig cells. Mibefradil was highly effective both in blocking the T type Ca2+ channels and in reducing the StAR gene expression in Leydig cells. The steroidogenesis in Leydig cells is stimulated by the Ca2+ entry through the T-type VDCC, which appears to mediate the hCG effect on the steroidogenesis in Leydig cells.

P-510 Protein expression in human granulosa cell subtypes A. Eldib1, E. Iammarone2, D.J. Gillott1, A.H. Handyside2, J.G. Grudzinskas2 1 St Bartholomews Hospital, Reproductive Physiology, London, UK; 2London Bridge Fertility Centre, Gynaecology and Genetics Centre, London, UK Introduction: Follicular development requires a series of complex interactions between oocyte and granulosa cells. The cumulus granulosa cells (cGC) are adjacent to and remain in intimate contact with the oocyte, while the mural granulosa cells (mGC) retain a greater spatial distance from the oocyte. Nevertheless, these germ and somatic cells maintain close association and reciprocally mediate the ordered series of maturation and differentiation steps required for oogenesis. These events are regulated by systemic factors that may come from the plasma, being produced locally by granulosa cells, or even by the oocyte itself. Further, gap junctions link cGC, mGC and the oocyte allowing the transfer of small regulatory molecules or metabolic precursors. The aim of this study is to establish an experimental model in order to investigate these events in vitro, and to distinguish sub-populations of granulosa cells at the molecular level. Materials and methods: GC were obtained from 12 women undergoing controlled ovarian hyperstimulation for IVF/ICSI and were isolated by centrifugation over percoll gradients prior to overnight culture, before washing to remove non-adherent cells. Cells from three patients at a time were pooled placed in 0.5 ml of culture medium (RPMI) without methionine, followed by the addition of L-[35S]-methionine (1 mCi/ml) for metabolic radiolabelling and a further 3 h incubation. All cells were washed and subjected to detergent lysis, before 2D electrophoresis was performed. The resulting 2D gels were dried and exposed to light sensitive phosphoimager plates for 10 days and scanned by a phosphoimager, exposed to X-ray film, or in the case of unlabelled cells, subjected to silver staining. Results: The mean number of separate protein spots subjected to semi-quantitive computerized analysis was 856 (±190) per gel. Neither constitutive nor biosynthetic proteomes resemble that of serum or any other known bodily fluid as described by the Swiss Institute of Bioinformatics (Geneva). At least six protein spots with molecular weight between 25 and 60 kDa with isoelectric points between pH 4.5 and 6 were identified as being unique to either cGC or mGC. These results show the constitutive and biosynthetic

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proteomes of human granulosa cells, and differences in protein expression and biosynthesis between these cell types are discussed. Conclusions: We believe that these data represent the first published biosynthetic proteome of sub-populations of human granulosa cells. The experimental techniques for the collection, culture and proteomic analysis that we have established may enable us to isolate sub-populations of granulosa cells from human follicles or correlate their protein expression with the eventual success or failure of fertilization and embryo development. In addition, this experimental model may be used to assess the molecular effects of known endocrine, autocrine and paracrine factors that may influence follicular development.

POSTER SESSION

Female fertility: clinical aspects P-511 A retrospective audit: Four years of follow up of the management and the reproductive outcome of the patients referred to a specialist infertility clinic M. De, S. Astley, K. Best, I. Aird Queen Elizabeth Hospital, Obstetrics and Gynaecology, Gateshead, UK Introduction: The aim of this retrospective cohort study was to follow up the management and the overall reproductive outcome of couples referred to an infertility clinic over a period of 1 year. Complications of pregnancies in women who conceived spontaneously or following ovulation induction with clomiphene (CC), IUI and IVF/ICSI treatment were also analysed. Limited information is available in the literature concerning follow up of couples with subfertility. Materials and method: One hundred and seventy-nine couples were identified from the outpatients appointment register as new patients referred to the specialist infertility clinics at QE Hospital, Gateshead in the year 2000. One hundred and seventy-one clinical records were reviewed by a hand held search. Data on female age at referral, initial investigations, final diagnosis, treatments offered, pregnany outcome, antenatal complications, mode of delivery, gestation at birth, birth weight and gender were recorded in 140 patients with subfertility issues. Result: The mean age of female partners was 30.6 years. Of the referred couples 17% did not have any investigation performed prior to referral. Tubal factor seems to be the leading cause (27%) of infertility followed by anovulation (21%), unexplained and male factor (17.5% each), multifactorial (8%), endometriosis (3%) and congenital problems (3%). Three couples had iatrogenic factor and one couple was in unisex relationship. Of the patients 17% became pregnant spontaneously within 4 years from referral. Twenty patients received 106 cycles of CC treatment with a cumulative live birth rate (CLBR) of 20% per woman or 5% per cycle. Sixteen couples received 51 cycles of IUI treatment which resulted in a CLBR of 19% per couple or 8% per cycle. Fifty-four couples had IVF/ICSI treatment over 90 cycles with a CLBR of 43% per woman or 26% per cycle. Two couples adopted children. Overall 38% of all referred couples with a fertility issue had a baby within 4 years of referral. When pregnancy outcomes were compared according to mode of conception (i.e. spontaneous, CC, IUI and IVF/ICSI); primiparous patients were found more likely to fall pregnant than multiparous patients except in CC pregnancy group. Mean birth weights seem to be lower in CC pregnancies. Antenatal complications, preterm labour, emergency section were highest in CC pregnancies followed by IVF/ICSI pregnancies. Male to female baby ratio was higher in IVF/ICSI and IUI treated group. Conclusion: The patients were referred in a relatively young age. 17% of couples were referred without any preliminary investigation. Tubal factor infertility seems to be a predominant factor. Almost one in six women are likely to fall pregnant spontaneously. Two out of five referred couples would have a baby within the next 4 years. Pregnancies from Clomiphene treatment are more at risks of antenatal complication, preterm births, low birth weight babies and emergency section.

Abstracts of the 21st Annual Meeting of the ESHRE, Copenhagen, Denmark, 19–22 June 2005

P-512 A reduction in intraovarian arterial blood flow resistance after ovulation is necessary to achieve pregnancy in natural cycle K. Nakagawa1, M. Sato1, M. Ito1, T. Horikawa1, W. Iwasaki1, S. Kawachiya2, E. Fujii2, Y. Takahashi1, H. Saito1 1 National Center for Child Health and Development, Division of Reproductive Medicine, Tokyo, Japan; 2National Center for Child Health and Development, Division of Gynecology, Tokyo, Japan Introduction: Color Doppler imaging permits the accurate localization of vessels and high-frequency pulsed Doppler ultrasonography has improved the resolution of flow velocity waveforms. It has been reported that intraovarian blood velocity increases approximately 29 h before the time of follicular rupture and that the mean values for intraovarian pulsatility index (PI) tend to be lower during the immediate periovulatory period. However, information concerning changes in intraovarian blood flow in relation to the outcome of infertility treatment is not available. In this study, intraovarian arterial blood flow before and after follicle rupture in the natural cycle was examined using transvaginal color flow Doppler imaging and changes in intraovarian arterial resistance in relation to the outcome of infertility treatment was analyzed. Materials and methods: In a prospective study, 227 spontaneous cycles in 118 infertile patients who were undergoing infertility treatment at the division of Reproductive Medicine in our center were recruited in this study. The impedance to flow in intraovarian vessels was measured by means of transvaginal color flow Doppler imaging during the periovulatory period in the natural cycle of all patients. The PI of intraovarian arterial blood flow and pregnancy rate was evaluated. Results: The increasing degree of intraovarian arterial PI values after follicular rupture compared with that before follicular rupture is defined as the PI increasing rate, and this rate was calculated as follows; the intraovarian arterial PI value after follicular rupture was divided by that before follicular rupture. According to the PI increasing rate, the 227 cycles could be classified into eight groups and the pregnancy rate in group A (whose PI increasing rate was 30% and more) was 4.5%. Pregnancy rates in group B (20%¼PI increasing rate

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