[Comparison of oral dydrogesterone with micronized vaginal progesterone in fresh embryo transfert in IVF/ICSI]. / Comparaison de la dydrogestérone avec une progestérone micronisée vaginale dans le transfert d'embryon frais en FIV/ICSI.

INTRODUCTION: Luteal insufficiency corresponds to a progesterone deficiency affecting women who receive treatment for in vitro fertilization (IVF). Different routes of progesterone administration exist and have varying degrees of acceptability to patients. The aim of this study was to compare two luteal phase support (LPS) treatments: oral dydrogesterone versus micronized vaginal progesterone on the clinical pregnancy occurrence after fresh embryo transfer. MATERIAL AND METHODS: This study was a monocentric historical and observational cohort study carried out in the reproductive medicine department at the University Hospital, Femme Mère Enfant in Lyon. All the data were collected retrospectively. Women between 18 and 43 years old, who completed an IVF cycle with or without ICSI, followed by fresh embryo transfer on the second or third day after oocyte retrieval (D2 or D3) or at the blastocyst stage (D5 or D6) between July 2019 and July 2020 were included. The 290 patients included between July 2019 and January 2020 received 600mg per day of PMV. The 290 patients in the OD group included between January and July 2020 received 30mg OD per day. RESULTS: In the univariate analysis, the clinical pregnancy occurrence per transfer was comparable between the MVP and OD groups (P>0.05) (OR [95% CI]): 0.904 [0.630 ; 1.296]. In the multivariate analysis, OD also appeared to be associated with a similar pregnancy occurrence compared to MVP, with a non-significant difference (OR [95% CI]): 0.940 [0.640; 1.380]. The use of OD compared to MVP did not significantly influence the clinical pregnancy occurrence in any age group. There was no significant difference between the two groups in the clinical pregnancy occurrence, whether the patients belonged to the reference population of the center or not (P>0.05) (OR [95% CI]): 2.367 [0.568; 3.568]. CONCLUSION: This important French retrospective study confirms the safety and efficacy of OD.

[Administering GnRH Agonists in the luteal phase of Artificial Cycle Frozen-Thawed Embryo Transfers. A prospective randomized study]. / Administration d'agonistes de la GnRH (Gonadotrophin Releasing Hormone) en phase lutéale des protocoles substitutifs de transferts d'embryons congelés: étude prospective randomisée.

OBJECTIVE: Adding GnRH agonists in the luteal phase has recently been said to improve implantation in IVF treatment (increased rates of pregnancy and birth). Adding GnRH agonists could also be beneficial for frozen-thawed embryo transfers. The objective was to compare the administration of Gonadotropin Releasing Hormone (GnRH) agonists during implantation with usual progesterone supplementation in the artificial cycle of frozen-thawed embryo transfers. METHODS: A prospective randomized controlled trial was conducted in a reproductive medicine center in a university hospital including all women starting an artificial cycle of Frozen-Thawed Embryo Transfers (FET). Two hundred and twenty women were randomized from September 2013 to June 2014. In the addition of GnRh agonists' group, two triptorelin injections of 0.1mg were carried out on the 4th day and on the 6th day following the introduction of progesterone. The primary outcome was the ongoing pregnancy rate. RESULTS: The ongoing pregnancy rate was higher (17 % versus 10.6 % P=0.29) when triptorelin was added, although the difference wasn't significant for the population as a whole. The increase proved to be significant in the case of day 2 embryos (34.6 % versus 10.3 % P<0.05) and of vitrified blastocysts (33.3% versus 12.5% P<0.05). CONCLUSION: The ongoing pregnancy rate for day 2 embryos and vitrified blastocysts significantly increased when GnRH agonists were added during implantation.

[Laparoscopic promontofixation: defining early morbidity using a standardized method]. / Promontofixation par cœlioscopie : évaluation de la morbidité précoce par une méthode standardisée.

OBJECTIVE: Adverse event reporting for laparoscopic promontofixation is highly variable and non-standardized in the literature. The aim of this study was to better characterize early postoperative complications of laparoscopic promontofixation for genital prolapse using a standardized reporting methodology. PATIENTS AND METHODS: A retrospective study was conducted on 174 women with genital prolapse undergoing laparoscopic promontofixation from January 2008 to January 2013. Complications arising during the first month after surgery were reviewed according to the Clavien and Dindo classification. RESULTS: At least one postoperative adverse event was reported in 57 out of 174 (33 %) women, grade 1 in 22 patients (13 %), grade 2 in 31 patients (18 %) and grade 3 in 4 patients (2 %). No patient experienced a grade 4 or 5 complication. Fifty-three out of 57 (93 %) complications were grade 1 or 2. The most frequently reported adverse event (n=24; 14 %) was constipation (grade 2). DISCUSSION AND CONCLUSION: Laparoscopic promontofixation is a safe procedure with almost exclusively benign (grade 1 or 2) early complications. The hypothesis of induction or increasing constipation by this type of genital prolapse surgery should be further evaluated.

Sexuality after laparoscopic Davydov in patients affected by congenital complete vaginal agenesis associated with uterine agenesis or hypoplasia.

BACKGROUND: Several surgical techniques have been described for the treatment of patients with vaginal agenesis. The simplest intervention that gives good sexual results should be the surgical technique of choice. METHODS: We report anatomic and functional outcome in 28 women after vaginoplasty using laparoscopic Davydov operation. This surgery includes three steps: two laparoscopic and one perineal. The patient then has to use a mould or a vaginal dilatator for 1 month. The functional outcome was assessed by a brief and valid self-report questionnaire evaluating female sexual life (Female Sexual Function Index, FSFI). A control group was recruited to compare the results. RESULTS: Two intra-operative bladder and ureteric injuries were repaired without sequels. Two post-operative complications were observed: one abdominal migration of the mould, which was treated successfully with the laparoscope, and one vesico-vaginal fistula. No patient was lost to follow-up. The anatomical result was judged to be satisfactory (>6 cm) in 26 of the 28 patients: the mean vaginal length was 7.2 +/- 1.5 cm. Among the 28 operated women, 19 had a good or very good result. No statistical difference was found between our operated patients and French controls in all six domains of the FSFI. CONCLUSIONS: Laparoscopic Davydov may be considered a good option for the surgical treatment of women presenting vaginal agenesis. This technique offers advantages such as: short operating time and hospital stay, no particular instrumentation required and no external scars. Sexuality approaches so-called 'normal sexuality'.

Effect of radiotherapy on pelvic lymph node metastasis in cervical cancer stages IB2-IVA: a retrospective analysis of two comparative series.

Efficiency of radiotherapy in controlling lymph node metastasis is a controversial issue. A continuous series of 87 patients affected by cervical cancer stages IB2-IVA and treated using pelvic radiotherapy is presented. A retrospective comparison is made between two populations. In the two populations, a staging lymphadenectomy was carried out before the onset of the therapeutic program. In the first population (53 patients), the pelvic nodes only were dissected and in the second one (34 patients), the pelvic lymph nodes were left in place and the paraaortic nodes only were dissected. In both series, a completion surgery was performed after finalization of the radiotherapy. It was carried out at open abdomen in both series. It included a systematic pelvic dissection for the patients whose pelvic nodes had been intentionally left in place at the time of the initial staging lymphadenectomy. Both series were identical as far as classic risk factors were concerned (FIGO stage, maximal tumor diameter, lymphovascular space involvement). The radiotherapy administered to the pelvis was the same in both populations. The number of patients with pelvic lymph node metastasis was 21 (39.6%) in the first population versus 6 (17.6%) in the second one (P = 0.03). The percentage of positive lymph nodes among the retrieved lymph nodes was 18.94 in the first population versus 2.8 in the second one (P = 0.0001). Pelvic radiotherapy is likely to control most of the pelvic lymph node metastasis, but not all of them. Practical deductions and further developments are discussed.

[Laparoscopic Davydov or laparoscopic transposition of the peritoneal colpopoeisis described by Davydov for the treatment of congenital vaginal agenesis: the technique and its evolution]. / Le Davydov coelioscopique ou transposition coelioscopique du colpopoiesis péritonéal pelvien décrit par Davydov pour le traitement des agénésies congénitales du vagin: la technique et son évolution.

INTRODUCTION: The laparoscopic Davydov is described. The data concerning the surgery and the postoperative course are reported at the same time as the data concerning the anatomical and sexological results. PATIENTS AND METHODS: The surgery includes three steps: (i) cleavage under laparoscopic guidance, (ii) peritoneovestibular stitch by perineal approach, (iii) making the vaginal vault with the laparoscope. The cleavage can be performed in front of the fascia interposed between the bladder and the rectum or behind it. A nymphoplasty can be added to the colpopoeisis. RESULTS: Between February 1996 and March 2003 we operated on 28 patients affected by congenital vaginal agenesis using the laparoscopic Davydov technique. Two peroperative complications occurred (urinary tract injuries during the first step: laparoscopic management) and two postoperative complications (intraperitoneal migration of the mould and vesicovaginal fistula managed successfully with the laparoscope for the first one and trough laparotomy for the second one). Four re-operations (incision and dilation) were necessary. The length of the neovagina was, at the last assessment, 7.2 +/- 1.3 cm. The Female Sexual Function Index was 26.5 +/- 5.6 vs. 27.9 +/- 4.5 in a control cohort. In the patients whose cleavage was performed behind the fascia (13 cases vs. 15) no complication occurred, no re-operation was necessary, the length of the neovagina was 7.0 +/- 0.7 cm and the FSFI was 26.3 +/- 5.9. DISCUSSION AND CONCLUSION: The laparoscopic Davydov is, if the dorsal approach is used for the cleavage, an easy to make operation (operating time: 90 +/- 29 minutes) with a short hospital stay. The postoperative care is simple (vaginal mould useless). Heterosexual activity with penile penetration can start early (6 to 8 weeks). The level of satisfaction is high. Laparoscopic Davydov procedure may be considered a good alternative to the more complex ones (as Vecchietti's technique) or to the more dangerous ones (sigmoid colpoplasty).

Reproducibility of transvaginal three-dimensional endometrial volume measurements with virtual organ computer-aided analysis (VOCAL) during ovarian stimulation.

OBJECTIVE: To calculate inter- and intraobserver reliabilities for three-dimensional endometrial volume measurements during in vitro fertilization using virtual organ computer-aided analysis (VOCAL). PATIENTS AND METHODS: Three-dimensional ultrasound measurements of the endometrium using VOCAL software were performed on the day of oocyte retrieval in each of 79 consecutive patients undergoing in vitro fertilization or intracytoplasmic sperm injection. Endometrial volume was calculated every 15 degrees and every 30 degrees by two different observers in order to determine the reproducibility of the technique. RESULTS: Intraobserver reliability for the 15 degrees measurements was 0.97 for both observers; for the 30 degrees measurements, it was 0.93 for one observer and 0.96 for the other. Interobserver reliability was 0.80 for the endometrial volumes calculated every 15 degrees and 0.83 for the volumes calculated every 30 degrees. The intra- and interobserver measurement agreement showed good reproducibility. However, the volumes calculated every 15 degrees were more accurate because the means of differences were close to zero. CONCLUSION: VOCAL provides a reproducible method for the estimation of the endometrial volume.

Neoadjuvant chemotherapy for unresectable ovarian carcinoma: a French multicenter study.

BACKGROUND: Initial debulking surgery followed by chemotherapy is the current treatment for International Federation of Gynecology and Obstetrics Stage IIIC/IV ovarian carcinoma but has a limited efficacy when optimal cytoreduction is not achieved at the end of the surgical procedure. An alternative treatment for these patients could be neoadjuvant chemotherapy. The purpose of this retrospective study was to report the results of neoadjuvant chemotherapy in operable patients (no medical contraindication to surgery) presenting with primary unresectable tumors. METHODS: Between January 1996 and March 1999, operable patients presenting with Stage IIIC or IV ovarian carcinoma underwent, in six French gynecologic oncology departments, surgical staging to evaluate tumor resectability. When the tumor was deemed unresectable by standard surgery, the patient received three to six cycles of platinum-based neoadjuvant chemotherapy according to the response and the center's usual protocol. Patients were surgically explored after completion of neoadjuvant chemotherapy when the tumor did not progress during treatment. Debulking was performed during this secondary surgery when a response to chemotherapy was observed. RESULTS: Fifty-four patients were treated by neoadjuvant chemotherapy. The first surgical staging procedure was laparoscopy in 33 patients (61%) and laparotomy in 21 patients (39%). The median number of neoadjuvant chemotherapy cycles was 4 (range, 0-6). Forty-three patients (80%) responded to neoadjuvant chemotherapy and then tumors were debulked. Optimal cytoreduction was obtained in 39 patients (91% of the patients who underwent debulking) and with standard surgery in 32 patients (82%). For patients whose tumors were optimally debulked, blood transfusions were administered to 17 patients (43%), median intensive care unit stay was 0 days (range, 0-7 days), and median postoperative hospital stay was 10 days (range, 4-62 days). Median overall survival for the total series was 22 months. Survival was better for patients debulked after neoadjuvant chemotherapy compared with patients with nondebulked tumors (P < 0.001). CONCLUSIONS: Neoadjuvant chemotherapy for primary unresectable ovarian carcinoma leads to the selection of a subset of patients sensitive to chemotherapy in whom optimal cytoreduction can be achieved after chemotherapy by standard surgery in a high proportion of cases. Conversely, aggressive surgery can be avoided in patients with initial chemoresistance, in whom the prognosis is known to be poor regardless of treatment.

Evaluation of global DNA hypomethylation in human colon cancer tissues by immunohistochemistry and image analysis.

BACKGROUND: Global hypomethylation of DNA is frequently observed in human tumours. This alteration is detected in early adenomas in colorectal tumorigenesis. Information is currently acquired after extraction of DNA from tissues, digestion with nucleases, and analysis by reverse phase chromatography, or treatment with restriction enzymes followed by gel electrophoresis analysis and Southern hybridisation with radiolabelled probes. AIMS: The purpose of our work was to evaluate the global methylation status of DNA in malignant lesions without loosing the histopathological features of the samples. PATIENTS: The investigation was performed on paired normal-tumour tissues from 13 patients undergoing surgical resection of colorectal adenocarcinomas. METHODS: Antibodies raised against 5-methylcytidine can be used to label methyl rich regions in interphase nuclei. This technique was adapted to the study of paraffin embedded tissues and an immunohistochemical method was developed to assess the global methylation status of individual nuclei while preserving cell morphology and tissue architecture. Computer assisted quantification of the staining intensity was performed on malignant and normal zones of human colon tissues to test the correlation between the immunolabelling signal and the respective histological patterns observed. RESULTS: Qualitative and quantitative differences were observed and measured between the normal and malignant part of each sample. Morphologically altered nuclei displayed densely labelled spots within faintly labelled areas whereas normal nuclei were darker and uniformly stained. Image analysis allowed calculation of the average integrated optical density of the nuclei in both types of tissues, demonstrating a constant and significantly lower intensity for the former type of cells.

Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl-CpG binding proteins. In cell lines and cancers, inhibition of tissue-specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5' end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of approximately 2.7 kb in length. In human sporadic breast cancers, the down-regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl-CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl-CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl-DNA binding proteins may participate in the regulation of gene expression in mammalian cells.

Proteasomes in human spermatozoa.

In the present study we describe the localization of proteasomes in human spermatozoa by means of immunolabelling with different monoclonal and polyclonal antibodies detected by confocal microscopy. Western blotting confirmed the specificity of the antibodies and has shown that proteasomes are present in spermatozoa and in seminal fluid. In spermatozoa proteasomes are concentrated in the neck region where the centrioles are located. Some labelling was also detected at the periphery of the head, but no proteasomal antigens were detected in either the nucleus or associated with the flagellum. Proteasome inhibitors did not affect the motility of the spermatozoa, acrosome reaction nor zona binding. It is hypothesized that paternal proteasomes enter the oocyte during fertilization in tight association with the centrioles and may serve a special function during further development which can be associated with the function of a hypothetical proteolysis centre.

Localization of proteasomes in human oocytes and preimplantation embryos.

In the present study we describe the localization of proteasomes in human oocytes, apoptotic preimplantation embryos, and triploid preimplantation embryos by means of immunolabelling with the MCP21 monoclonal antibody detected by confocal microscopy. While in the oocytes proteasomes are scattered throughout the cytoplasm, in the pronuclear zygote they appear to concentrate at the periphery of the cytoplasm and do not enter the pronuclei. During early cleavage stages, proteasome immunolabelling is concentrated in the nuclei, while the examination of triploid blastocysts showed that proteasomes had a similar cellular distribution to somatic cell lines, i.e. in the nuclei but not in the nucleoli or the cytoplasm. It appears that the distribution of proteasomes dramatically changes during human preimplantation embryo development.

Measurement accuracy in confocal microscopy.

Confocal laser scanning microscopy provides optical serial sections through thick biological samples, making it possible to perform both three-dimensional visualization and three-dimensional quantitative analysis. On human lymphocytes, we measured geometrical features, cell contents in DNA and in cyclin A and CDK1 proteins, localization and colocalization of these two proteins. Cells were acquired at a vertical sampling step of 0.5 micron, which gives sufficient information about cell labelling. For the purpose of obtaining fast and reliable data at a reduced time cost, we examined various possibilities to simplify acquisition. For example, it might be possible to increase the vertical sampling step to 2.0 microns while preserving an acceptable accuracy of measurements. Further limiting the acquisition to the central sections appeared to give only rough estimations about the whole cells. Finally, we compared confocal microscopy to conventional two-dimensional epifluorescence microscopy. Confocal microscopy appeared slightly less accurate as regards content estimation, but was an invaluable tool when investigating three-dimensional structures and, more especially, localization of proteins.

Effect of gastrin-releasing peptide on sperm functions.

Male infertility can be related to defects in motility, capacitation, acrosome reaction, binding and penetration of the zona pellucida. While different in-vitro techniques (such as micromanipulation which is complicated and expensive) are available for the treatment of male infertility, several pharmacological agents have been shown to increase fertilizing capacity under accurate experimental conditions. Gastrin-releasing peptide (GRP, the mammalian homologue of the amphibian skin peptide bombesin) is present in the reproductive tract and expressed by the pregnant ovine endometrium prior to attachment and throughout the pregnancy. A bombesin-like peptide resulting from alternate splicing of the GRP gene in testis has been detected in primates. In this study, we have tested the ability of GRP to enhance human sperm functions such as motility, capacitation, zona binding and acrosome reaction. Analysis of sperm motility was performed with the ATS 20 computer-assisted semen analysis (CASA) system. Zona binding was analysed using intact human unfertilized oocytes and selective labelling of spermatozoa with two fluorochromes. Our results did not show any positive effect of GRP on these parameters under our experimental conditions. However, when GRP at the concentration of 100 nM was added after ionophore treatment, the percentage of reacted cells increased. significantly (P < 0.05) compared with situations where each agent was used alone. This led us to suppose that the role of bombesin in the different stages of fertilization might not exclude other unknown factors.

Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy.

Fluorescein isothiocyanate (FITC) is largely used in immunofluorescence methods. We propose to analyse the quality of some recent fluorochromes using image analysis. Fluorochromes tested include FITC and dichlorotriazinylaminofluorescein (DTAF), dipyrrometheneboron difluoride (BODIPY), Rhodol Green and cyanine 2. RAMOS cells were immunolabelled against the proliferating cell nuclear antigen (PCNA) revealed by the biotin-streptavidin technique. Slides were mounted in anhydrous glycerol or in buffered glycerol (pH 7.0 or pH 8.5). No antifading medium was added. Cell fluorescence emission intensity and bleaching characteristics were measured. Rhodol Green exhibited the highest fluorescence intensity and the best photobleaching resistance. Although BODIPY also resisted well during the photobleaching assay, its fluorescence intensity was weak. FITC, DTAF and cyanine 2 showed intermediate fluorescence intensity and a fast decay of fluorescence. Among the green emitting fluorochromes tested, Rhodol Green appeared to be the best.

n-Butyrate, a cell cycle blocker, inhibits early amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of duck hepatocytes.

During chronic hepadnavirus infection, virus persistence depends on the regulation of the pool of covalently closed circular DNA (cccDNA), which is the template for transcription of viral RNA species. The development of in vitro infection of duck hepatocyte primary cultures by duck hepatitis B virus (DHBV) provides a unique opportunity to study the regulation of cccDNA synthesis. After DHBV in vitro infection, cccDNA is detected 1 day later and is amplified to a high copy number after 1 week in culture. We studied whether this amplification occurs during cell cycle progression of duckling hepatocytes. By using [3H]thymidine incorporation, we found that hepatocytes obtained from 3-week-old ducklings spontaneously entered the S phase of the cell cycle when cultured in serum-free medium without added growth factors. Bromodeoxyuridine labeling confirmed that cellular DNA synthesis took place in more than 50% of parenchymal cells. Cytofluorometry analysis revealed the presence of asynchronous populations and polyploidization processes. The addition of a cell cycle blocker, n-butyrate, completely inhibited [3H]thymidine incorporation and blocked duckling hepatocytes in the G1 phase of the cell cycle. Simultaneously, butyrate inhibited cccDNA amplification and allowed the establishment of DHBV infection, as demonstrated by the detection of a basal level of cccDNA in treated hepatocytes. Both effects were reversible since active cell DNA synthesis was restored and cccDNA accumulated after drug withdrawal.

Effects of progesterone on human spermatozoa prepared for in-vitro fertilization.

Progesterone has been tested in vitro with human spermatozoa to verify its physiological effects and its possible therapeutic use in cases of male infertility. Progesterone induced a rapid, dose-dependent influx of calcium in capacitated and non-capacitated spermatozoa with a half-maximally effective dose of 30 nM. The agonist, 19-nortestosterone, was much less potent that progesterone itself. Progesterone-induced calcium influx was not inhibited by a similar concentration (0.1 microgram/ml) of RU 486, a classical progesterone antagonist. The increase in intracytoplasmic calcium levels was unable to induce the acrosome reaction (AR) even after incubation for 5 h, when this was evaluated by double staining, using a monoclonal antibody GB24 raised against the inner acrosome membrane and ethidium homodimer as a vital probe. However, after incubation for 5 h, progesterone was able to enhance the tyrosine phosphorylation of a 95 kD sperm protein, which is phosphorylated progressively during capacitation in well-defined culture media. Incubation of spermatozoa with 1 or 10 micrograms/ml progesterone for 3 or 30 min did not induce major modifications of hyperactivated movement when analysed by computer-assisted semen analysis. Progesterone secreted by cumulus cells may physiologically increase sperm intracytoplasmic free calcium during capacitation. This priming effect may facilitate the acrosome reaction, induced by binding to the zona pellucida, without enhancing spontaneous acrosome reaction prematurely. It therefore seems useful to propose progesterone as a means of accelerating capacitation during in vitro fertilization in cases of male infertility.

Quantitative immunochemistry of endothelial cells in cutaneous tissue.

In vivo endothelial cell exploration in human pathology has been limited by the lack of a suitable procedure for quantification of endothelial molecule expression. The objective of the present study was the development of a precise, simple and rapid image analysis method to quantify dermis endothelial cells and associated molecules. Cutaneous tissue sections were labelled by immunoperoxidase procedure using a monoclonal anti-von Willebrand factor (vWF) antibody, which identifies endothelial cells. The image analysis programme was tested to quantify final reaction deposit (DAB) with reference to different anti-vWF antibody concentrations. The entire biopsy section was acquired, field by field. Analysis was restricted to three dermis zones geodesically defined from epidermis. Optical density and area measurements were performed in whole area and in positive areas. This work demonstrated the feasibility of endothelial cell study assisted by image analysis. Surface labelled by anti-vWF antibody at saturating concentration may be considered as an endothelial reference surface, and thus may be useful for future quantification of other endothelial molecules.

Expression of cell cycle regulatory proteins in chronic lymphocytic leukemias. Comparison with non-Hodgkin's lymphomas and non-neoplastic lymphoid tissue.

The expression of certain cell cycle regulatory proteins: cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin E, Bcl2 and PCNA was examined in peripheral blood lymphocytes (PBL) from 25 cases of chronic lymphocytic leukemias (CLL) in order to analyze a possible cell cycle involvement of CLL lymphocytes. For comparison, we also studied the expression of these proteins in: 23 samples of non-Hodgkin's lymphoma (NHL) tissue of different histological types, 10 samples of non-neoplastic lymphoid tissue (NLT), non-stimulated PBL (NS-PBL) and PHA-stimulated PBL (PHA-PBL) from three healthy donors. Samples were lysed and proteins were resolved on polyacrylamide gel followed by Western blot. The expression of cdk4 and cyclin E, both known to act in early cell cycle stage, was approximately on the same level in all groups of lymphoid pathology examined. In particular, we found that that 19 out of 24 CLL cases were cyclin E positive and all but one were cdk4 positive, ie they expressed these markers over twice the level of non-stimulated healthy PBL. The cdk1 expression was above the level seen in NS-PBL in 14 (56%) cases, but the average expression was significantly lower than in the other tissues examined, including low-grade lymphomas. Cdk2 expression was comparable in CLL and in low malignancy grade NHL, but weaker than in other NHL and in NLT. Cyclins A and B, normally observed in advanced cell cycle phases, were not seen in any CLL case. The presence of cdk4 and cyclin E in the blood cells of the majority of CLL cases studied, as well as cdk1 and cdk2 in some cases, indicate that the CLL cells are not quiescent, but are blocked in an early stage of the G1 cell cycle phase, and/or that the expression of these proteins is pathologically deregulated.

Methods for cell proliferation analysis by fluorescent image cytometry.

Methods were developed for multimodal microscopic image analysis in order to identify and analyze one cell type under various microscopic conditions. Our purpose was to quantify both total DNA content using propidium iodide (PI) stain and S-phase fraction using the bromodeoxyuridine (BrdUrd) incorporation technique in cell population subsets. The model chosen was plasma cells in bone marrow triply labelled with fluorescein isothiocyanate (FITC) for intracytoplasmic immunoglobulins, with amino-methylcoumarin-acetate (AMCA) for BrdUrd, and with PI for DNA. Image analysis included three phases. First, plasma cells were recognized on FITC images, and the centroid positions were stored. Second, plasma cell nuclei were geodesically reconstructed from these stored positions using PI images in which DNA content was measured, and the nuclear mask outlines were stored. Third, BrdUrd incorporation level of plasma cells was measured on AMCA images inside PI nuclei masks and stored. Image DNA vs. BrdUrd scatterplots were obtained for cells selected according to the expression of intracytoplasmic immunoglobulin. Thus, both ploidy and proliferation could be independently evaluated on a subset of the cellular population.