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A total of 304 human pronuclear zygotes and cleaved embryos from the 2- to 9-cell stages, obtained during invitro fertilization attempts, were photographed and retrospectively analysed after transfer for their morphology and size in relation to their developmental stage, using the Imagenia programme of a Biocom 500 image analyser. Morphometric parameters were calculated from the perimeters, surface measurements, theoretical diameters and circularity factors for the different structures analysed. This report provides the morphometric characteristics of living embryos. For the whole population the mean values were: 157.4 microns for the external zona pellucida diameter, 121.8 microns for the internal zona pellucida diameter, 17.9 microns for the thickness of the zona pellucida and 117.2 microns for the embryo cell mass diameter. The morphometric characteristics of the pronuclear-stage population were significantly different from the cleaved cell stages. If the zona pellucida and cell mass embryo diameters increased slowly from the 2- to 9-cell stages, embryonic external diameters were higher and zona pellucida thicknesses were lower in odd than even number blastomere embryos. Preliminary results show that in cases where implantation occurs, the embryo has a lower zona pellucida thickness. A comparison of the different embryo cell stages confirmed the existence of an asynchronous division process during early embryo development. Global results show no evidence of morphometric differences between subpopulations of the embryos according to their microscopic grading. Deviations from the normal asynchronous division process, however, appear to be a new parameter to take into account during embryo scoring.(ABSTRACT TRUNCATED AT 250 WORDS)

Because the down-regulation by progesterone of cystic fibrosis transmembrane conductance regulator (CFTR) expression could be a useful specific marker to define the state of implant receptivity in endometrium, a competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed for quantifying the CFTR mRNA concentration in human endometrial samples. A competitor RNA was constructed with the same sequence as the CFTR sequence except for a 20-nucleotide insertion in the middle. The amplified products were separated by polyacrylamide gel electrophoresis. The ratio of CFTR band areas to competitor band areas provided the basis of quantification. Using this competitive RT-PCR, we measured CFTR mRNA in human endometrial samples taken at different periods of the menstrual cycle, in endometriosis, and in hyperplasia. Results show that the method is suitable for measuring the concentration of CFTR mRNA.

Objective: The aim of our study was to determine if the assessment of pulmonary vascularization by energy color Doppler during ultrasound examination can predict the absence of pulmonary hypoplasia before birth in situations where it is a high risk. Methods: In a prospective study of 12 pregnancies presenting a risk of pulmonary hypoplasia (5 early and prolonged premature ruptures of the membranes, 1 diaphragmatic hernia, 1 chylothorax, 1 pulmonary sequestration, 1 omphalocele, 1 anamnios and 2 Potter’s syndromes) energy color Doppler was used to visualize pulmonary vascularization. Results: In 10 cases where pulmonary vascularization could be visualized, none of the infants had pulmonary hypoplasia. In the 2 cases of Potter’s syndrome where pulmonary vascularization was not visualized there was a pulmonary hypoplasia. Conclusion: The visualization of fetal pulmonary vascularization with energy color Doppler in situations with a high risk of pulmonary hypoplasia can predict the absence of severe pulmonary hypoplasia.

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We investigated the activity of estrone sulfatase in normal and pathological endometrium. In normal endometrium, the estrone sulfatase activity [pmol E1 X min-1 X (mg prot)-1] was 23.13 +/- 8.44 (mean +/- SD). An increase (p less than 0.01) of estrone sulfatase activity (62.81 +/- 21.97) was noted in mild endometrial hyperplasia. In focal hyperplasia (when the measurements were performed in the normal endometrial biopsies) such an increase was not noted (19.10 +/- 5.33). Estrone sulfatase activities of moderate hyperplasia (25.30 +/- 11.40) and endometrial neoplasia (30.30 +/- 9.57) were in the same range as in normal endometrium. Treatment with progestagen simultaneously reduced hyperplasia and estrone sulfatase activity. But when morphologically abnormal endometrium persisted after treatment, estrone sulfatase activity remained increased. The increase of estrone sulfatase activity appeared to be specific to mild endometrial hyperplasia. The role of estrone sulfatase in the pathogenesis of endometrial hyperplasia is discussed.

4482 One major drawback of myeloablative allogeneic stem cell transplantation is the near 100% risk of profound fertility impairment leading to early menopause and sterility in most female patients. This toxic effect is especially critical in patients without malignant disease, i.e hemoglobinopathies or other inherited diseases. Cryopreservation of ovarian tissue with subsequent autotransplantation is an emerging procedure to preserve the fertility of young patients with a high risk of premature ovarian failure resulting from radiotherapy or chemotherapy for cancer. We report the restoration of ovarian activity and pregnancy with live birth after an orthotopic transplantation of frozen/thawed ovarian tissue in a 23 year old female patient given a transplant for sickle cell disease. In spring 2005, the patient of S/S hemoglobin genotype experienced a central nervous system stroke with unilateral central retinal artery occlusion. Monthly exchange transfusions were started from then and a pretransplant procedure was undertaken with a 10/10 matched sibling male donor who was heterozygous for HbS. In autumn 2005, biopsy samples of ovarian tissue were cryopreserved prior to conditioning. The patient received a conditioning regimen with IV busulfan (12.8 mg/kg total dose), cyclophosphamide (200 mg/kg total dose) and antilymphocyte globulin (15 mg/kg total dose) followed by allogeneic bone marrow transplantation (BMT). A stable 100% donor chimerism was obtained from D60.The patient developed grade II acute GVHD treated by steroids, then followed by limited chronic GVHD treated by a combination of mycophenolate-mofetil and ciclosporin. The treatment was tapered and stopped 7 months after BMT. After BMT, the patient presented clinical menopause with amenorrhoea and hot flushes due to premature ovarian failure. FSH and LH concentrations rose to menopausal levels. Hormone replacement therapy with oestro-progestogens was started 6 months after BMT and was maintained until ovarian function restoration after ovarian transplantation. In spring 2008, as the patient had been menopausal for 2.5 years as a result of the conditioning chemotherapy, an orthotopic autotransplantation of ovarian cortex was performed. The cortical fragments were grafted according to the following procedure: a first laparoscopy was performed to create a peritoneal windows between the right iliac vessels and an ovarian window in the remaining left ovary with the aim of further inducing angiogenesis. One thawed strip of ovarian cortex was cut in fragments, that were fixed in the ovarian incision and deposited in the peritoneal window. Three days later, a second laparoscopy was performed and three thawed cortical strips were fixed into the left ovary and one into the peritoneal window. Ovarian function recovery was evaluated by hormonal levels and follicular development was observed by ultrasound. The patient conceived spontaneously in a natural cycle in autumn 2008, and delivered a healthy female child in June 2009. This first case report of pregnancy and delivery after ovarian autograft in a patient treated by allogeneic BMT suggests that cryopreservation of ovarian tissue should be offered prior to SCT not only to women of childbearing age but also to prepubertal patients, as primordial follicles are already present in the post natal ovaries. This technique still deserves further investigations in leukemias but appears safe in non malignant diseases. Time line of treatment Disclosures: No relevant conflicts of interest to declare.