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Biomedical subjects

C Poirot

Publications and source records attributed to C Poirot.

18 recordsLinked to original sources

Multiparameter assessment of mouse oogenesis during follicular growth in vitro.

Comparison of oocyte development within the follicle in vitro and in vivo has a major impact on research into ovarian physiology and clinical practice. Despite major differences in ovarian physiology between rodents and humans, mice provide a useful model for studies of the endocrine and paracrine mechanisms controlling follicular development. In this study, early preantral follicles were isolated from 12-day-old mice and cultured individually in microdrops under oil during 6, 9 or 12 days. Taking into account previous observations, several oocyte criteria (diameter, chromatin configuration, transcriptional activity, intracytoplasmic calcium signalling and ability to undergo meiosis) were assessed to check that the development pattern of oocytes during follicle growth in vitro was similar to that already observed for oocytes developing in vivo, and that they reached the fertilizable oocyte stage. Results indicate that, during the 12-day-culture period, the oocytes grew until 74.3 +/- 4.2 microm, they became transcriptionally quiescent with a surrounded nucleolus (SN) chromatin organization, 50% of them exhibited regular calcium signals and 73.4% of them resumed meiosis. These data demonstrate that the protocol used generates oocytes with characteristics similar to oocytes allowed to mature fully in vivo and that it could be useful to set up the experimental culture of human ovarian follicles.

Animals↗

[Ovarian tissue cryopreservation for prepubertal girls: indications and feasibility].

OBJECTIVE: Survival improvement of children, adolescents and young women with cancer has led to consider with more cautiousness the long time iatrogenic side effects of treatments. Among those, premature ovarian failure has been described even for children. The aim of the study was to evaluate the indications and the feasibility of ovarian tissue cryopreservation for prepubertal girls. PATIENTS AND METHODS: From September 2000 to December 2004, 47 prepubertal girls were referred by oncologists for ovarian tissue cryopreservation. After informed consent, the ovarian tissue was collected and frozen by a slow cooling protocol until the temperature of liquid nitrogen. A histological analysis and a follicular account were performed. RESULTS: The harvest of ovarian tissue was performed for 45 patients. No surgical side effect occurred. The younger girls had a follicular density higher than the older. No metastatic ovarian tumour was found. DISCUSSION AND CONCLUSIONS: Numerous arguments as the follicular density in the ovary, the age of the patient, no surgical side effect, no metastatic ovarian tumour and recent progress in term of birth after ovarian tissue autografting allowed to think it is very important and ethical to propose an ovarian tissue cryopreservation even for children before sterilising treatment.

Adolescent↗

[Oocytes in vitro maturation: results and future in humans].

Mature oocytes are rare and highly specialized cells. In vitro maturation of human oocytes is an emerging assisted reproductive technology allowing to produce more mature oocytes without ovarian stimulation. Whereas in vitro maturation is technically more demanding than conventional in vitro fertilization for the laboratory, it carries many potential advantages, for example, in terms of lower treatment heaviness and removal of risk of severe ovarian hyperstimulation syndrome for the patients. Although the technology is still experimental, oocytes in vitro maturation has been successfully used and pregnancies and live births have been reported. Despite these successes, the overall efficiency of in vitro maturation remains low and this procedure must still be improved. The different steps of in vitro maturation process are shown and discussed as well as results in terms of pregnancy and live birth rates.

Cell Culture Techniques↗

[Cryopreservation of ovarian tissue].

Ovarian tissue cryopreservation (OTCP) is a new procedure of medically assisted procreation, still at the experimental stage, whose primary aim is to store female gametes as sperm cryopreservation permits to do for male gametes. Ovarian tissue is removed very simply by laparoscopy. It survives well to freezing if the medium contains a cryoprotective agent and the rate of freezing is slow. In contrast, thawing must be rapid. There are three processes for the utilization of ovarian tissue after thawing. In vitro maturation and xenografting remain impossible for technical and ethical reasons. Autologous transplantation (orthotopic or heterotopic) of the tissue is therefore the only foreseeable method over the short term. Indications for OTCP must remain rare as long as no pregnancy has been obtained in human. At the present time, only female patients who would inevitably suffer the loss of their fertility should be able to take advantage of OTCP. Basically, this would mean women subjected to castrating anticancer therapy. It would seem reasonable to set the age limit at 35-years for carrying out OTCP. Lastly, female patients should be clearly informed that the method is still at the research stage, and in France samples must be taken in accordance with the laws governing clinical research.

Adult↗

[Female fertility preservation before sterilizing treatment: contribution of ovarian tissue cryopreservation].

Longer survival after anticancer treatment has lead to concern about the long-term adverse effects. Altered fertility is of particular importance. Before sterilizing treatment, three non-exclusive methods can be proposed to preserve female fertility: in vitro fertilization followed by cryopreservation of embryos, cryopreservation of mature ovocytes, cryopreservation of ovarian tissue. The method or methods chosen will depend on the age of the patient, here marital status, the urgency of the treatment, and the type of disease. Embryo cryopreservation is a routine practice in medically assisted reproduction centers, while cryopreservation of mature ovocytes and ovarian tissue is still in the experimental phase. It is known however that mature ovocytes can be used after cryopreservation. Cyropreservation of ovarian tissue is a more difficult problem. To date, there have not been any pregnancies or births after freezing-thawing of human ovarian tissue. This tissue could be used in two ways: autograft and in vitro folliculo-ovocyte maturation. Despite the uncertainty concerning use, women cryopreservation of ovarian tissue quite well.

Cryopreservation↗

Effect of in-utero diethylstilboestrol exposure on human oocyte quality and fertilization in a programme of in-vitro fertilization.

Genital tract abnormalities and adverse pregnancy outcome are well known in women exposed in utero to diethylstilboestrol (DES). Data about adverse reproductive performance in women exposed to DES have been published, including controversial reports of menstrual dysfunction, poor responses after ovarian stimulation, oocyte maturation and fertilization abnormalities. We compared oocyte quality, in-vitro fertilization results and embryo quality for women exposed in utero to DES with a control group. Between 1989 and 1996, 56 DES-exposed women who had 125 in-vitro fertilization (IVF) attempts were retrospectively compared to a control group of 45 women with tubal disease, who underwent 73 IVF attempts. Couples suffering from male infertility were excluded. The parameters compared were oocyte quality (maturation abnormalities, immature oocyte, mature oocyte), fertilization and cleavage rate (per treated and metaphase II oocytes), and embryo quality (number and grade). We found no significant difference in oocyte maturational status, fertilization rates, cleavage rates, embryo quality and development between DES-exposed subjects and control subjects. These results suggest that in-utero exposure to DES has no significant influence on oocyte quality and fertilization ability as judged during IVF attempts.

Adult↗

[Intracytoplasmic sperm injection (ICSI) in the management of azoospermia].

OBJECTIVES: The objective of this study was to analyse all attempts of ICSI performed in our centre between 1995 and 1997 with surgically harvested sperm for the treatment of secretory and excretory azoospermia. MATERIAL AND METHODS: 71 infertile couples, in which the man suffered from secretory (n = 8) or excretory azoospermia (n = 63), were managed with 94 attempts of ICSI. RESULTS: ICSI was able to be performed 85 times with 82 embryo transfers resulting in 29 clinical pregnancies. The clinical pregnancy rate per ICSI attempt was comparable with sperm obtained on the day of ICSI or previously harvested and frozen (30.5% and 31.4% respectively). The clinical pregnancy rate per ICSI attempt was 33.3% for testicular sperm (n = 18), and 30.3% for epididymal sperm (n = 76). Twenty-seven clinical pregnancies (32.1%) were obtained for men with excretory azoospermia (47.2% for vas deferens agenesis and 20.8% for acquired urinary tract obstruction, p = 0.01), and 2 (20%) for men with secretory azoospermia. CONCLUSION: ICSI can achieve pregnancy in infertile couples in whom no other solution can be considered. Cryopreservation allows surgical harvesting to be dissociated from ICSI without decreasing the pregnancy rate, justifying freezing of sperm during any surgical procedure on the seminal tract. The better results obtained in congenital excretory azoospermia compared to acquired obstructions, usually post-infectious, suggest a harmful effect of infection on the quality of sperm.

Adult↗

Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions.

Numerous gap junctions exist between granulosa cells, between cumulus cells and between cumulus cells and the oocyte. They may play a role in the regulation of both follicular development and oocyte status. We used primary cultures of human granulosa cells to study the molecular nature and functionality of these gap junctions. As shown by a cinemicrographic technique, during the first 3 days of culture, cells flattened and extended in several directions by means of cytoplasmic extensions. An ultrastructural study showed the presence of both intercellular and annular gap junctions after 48 h of culture. As revealed by immunodetection analyses, connexin 43 was present. An analysis using a functional procedure, the gap fluorescence recovery after photobleaching (FRAP) method, indicated that: (i) diffusional communication existed among granulosa cells; (ii) the communication was delayed by treatment with 1-heptanol, a well-documented inhibitor of gap junction permeability; and (iii) permeability was up-regulated by incubation with 8-Br-cAMP, an analogue of cyclic AMP. The detection of connexin 43 and functional gap junctions in networks of cytoplasmic extensions indicated junction formation among cells during culture. In conclusion, our results show that human granulosa cells in culture exhibited functional gap junctions. Connexin 43 was present and the permeability of the gap junctions was up-regulated by cyclic AMP, an important modulator of human granulosa cell function.

Alcohols↗

Quantification of HIV-1 virus load under zidovudine therapy in patients with symptomatic HIV infection: relation to disease progression.

OBJECTIVE: To measure changes in HIV-1 virus load following zidovudine therapy, and to investigate the relationship between these changes and clinical progression. DESIGN: Prospective study of 18 symptomatic, zidovudine-naive patients, with CD4 count < 350 x 10(6)/l. METHODS: The following parameters were measured at each visit, before zidovudine therapy, after 1 month of therapy, and every 3 months thereafter. HIV-1 virus load in peripheral blood was determined by serum immune complex-dissociated HIV-1 p24 antigen (ICD-p24 Ag), quantitative plasma and cellular viraemia. A virologic response under zidovudine was defined as > 50% decrease in ICD-p24 Ag levels or > 1 log10 decrease in plasma or cellular viraemia titres from baseline values. CD4 and CD8 cell counts, and beta 2-microglobulin levels were also measured. Disease progression was defined as the time to a new AIDS-defining event or death. RESULTS: At enrolment, 13 out of 18 (72%) patients had positive ICD-p24 Ag and positive plasma viraemia, with a mean of 44 median tissue culture infective dose (TCID50) per ml; all patients had positive cellular viraemia with a mean TCID50 of 230 per 10(6)/l cells. Median CD4 cell count was 43 x 10(6)/l. Ten patients developed a new AIDS-defining event and eight died during a median follow-up of 15 months on zidovudine. Baseline prognostic markers for development of a new AIDS-defining event included ICD-p24 Ag, CD4 and CD8 cell counts, but only CD4 cell count remained predictive on multivariate analysis (P = 0.003). When each laboratory marker was analysed as a time-dependent covariate, only CD4 (P = 0.002) and CD8 (P = 0.001) cell counts predicted the occurrence of a new AIDS-defining event. Eight out of 13 (61.5%) patients had an ICD-p24 Ag response, and seven out of 13 (54%) a plasma viraemia response, but only cellular viraemia responders (five out of 18; 28%) had a 5.6-fold decrease in their risk of developing an AIDS-defining event (90% confidence interval, 1-33; P = 0.05). None of these markers correlated with survival. CONCLUSIONS: Plasma viraemia and ICD-p24 Ag, while providing useful short-term markers of zidovudine antiviral activity in vivo, do not correlate with disease progression in patients with advanced HIV infection. CD4 cell count remained the best initial and time-dependent predictor for development of new AIDS-defining events. Interestingly, a high CD8 cell count and a decrease in cellular viraemia titres also appear to be predictive of improved clinical outcome in this population.

Adult↗

Interaction between human immunodeficiency virus and Toxoplasma gondii replication in dually infected monocytoid cells.

THP-1 monocytoid cells, either not infected or chronically infected with human immunodeficiency virus type 1 (HIV-1), were challenged with Toxoplasma gondii. Parasitic growth, as assessed by trophozoite counting and measurement of supernatant p30 membrane antigen, was similar in HIV-infected and noninfected THP-1 cells. Also, T. gondii did not affect HIV replication. These experiments therefore failed to demonstrate any interaction between HIV-1 and T. gondii replication in concurrently infected monocytoid cells.

Animals↗

In vitro fertilization without ovarian stimulation: a simplified protocol applied in 80 cycles.

Ovulation induction with various hormonal agents has become a standard component of in vitro fertilization (IVF) cycles to obtain multiple oocytes. Failure to anticipate the retrieval of more than two oocytes often results in cancellation of the cycle. In this study, we report our results in 80 unstimulated IVF cycles. Serum estradiol (E2) and pelvic ultra-sound monitoring were begun on day 9 of the cycle. Human chorionic gonadotropin (hCG) was administered when the E2 level exceeded 180 pg/mL and the dominant follicle was greater than 18 mm. Eighteen pregnancies were obtained (22.5%/cycle), and 14 (17.5%/cycle) are ongoing. We conclude that favorable results can be obtained from unstimulated IVF cycles, despite replacement of a single embryo.

Adult↗

A new in vitro fertilization technique: intravaginal culture.

Intravaginal culture (IVC) is a new technique elaborated by the authors for the fertilization and culture of human oocytes. Its principle consists of fertilization and early development of the eggs in a closed, air-free milieu without the addition of CO2. One to five ovocytes are deposited in a tube completely filled with 3 ml of culture medium less than 1 hour after their recovery, with 10,000 to 20,000 spermatozoa per ml previously prepared. The tube is then hermetically closed and it is placed in the maternal vagina and held by a diaphragm for incubation for 44 to 50 hours. After this time, the content of the tube is examined and embryos are transferred to the uterus. In the first 100 consecutive punctures, 22 clinical pregnancies were obtained: 17 deliveries, 3 spontaneous abortions, and 2 tubal pregnancies. Also, a randomized study comparing IVC to in vitro fertilization (IVF) was done (160 cycles) and no statistically different cleavage, transfer, or pregnancy rate was seen between IVC and IVF. By simplifying the laboratory manipulations, this technique decreases the cost of IVF and permits its standardization and diffusion. It creates a psychologic comfort permitting active participation of the mother in this stage of embryo development. Also, the use of this technique may give greater knowledge of human gamete metabolism and of the physiology of reproduction.

Adult↗

Blood viscosity after a 1-h submaximal exercise with and without drinking.

Ten healthy subjects performed two exercise sessions similar to an endurance training session for average athletes (1 cycling exercise at 85% of the maximal heart rate on a Monark cycle ergometer). In the first session, the subjects were not allowed to drink during exercise. During the second session, the same subjects performed the same exercise as during the first session but they drank a beverage volume equal to the weight loss induced by the first exercise session to verify the hypothesis that the compensation of sweat loss by drinking could attenuate the exercise-induced blood viscosity increase. Both protocols (with and without drinking) induced a significant increase of hematocrit and plasma viscosity. The whole blood viscosity increased at all shear rates but this increase was significant only for the exercise protocol without drinking. Blood thixotropic property, erythrocyte deformability, and erythrocyte aggregability remained unchanged after both exercise protocols. Hemoconcentration explained the increase of hematocrit and plasma viscosity. Hemoconcentration was probably the consequence of a filtration process through capillary leakage in addition to sweat loss, which could explain the partial effect of drinking in our study.

Adult↗

[Intravaginal culture and embryo transfer].

The intra-vaginal culture and embryo transfer is based on the principle of fertilization of hyman ovocytes in the absence of CO2-enriched air. Ovocytes and spermatozoa are placed in a water-tight tube filled with B2 medium and placed in the vagina during the culture. Once the culture is done, the embryos are replaced in the uterus. The various studies done with intra-vaginal culture and embryo transfer, their results as well as future prospects are reviewed in detail.

Cells, Cultured↗

Human seminal fibrinolytic activity: specific determinations of tissue plasminogen activator and urokinase.

The determination of total fibrinolytic activity of ejaculates was realized by fibrin plate method. For the same seminal samples, tissue plasminogen activator (t-PA), urinary plasminogen activator (uPA) antigens and uPA activity were specifically quantified. The seminal values were fifty times higher than in the blood for t-PA and fifteen times for uPA. There was no correlation between the both levels but from split ejaculates measurements a higher concentration was observed in all first fractions. By zymography assays, it was shown that seminal plasminogen activators are under active forms. The lack of proUrokinase in semen was also demonstrated.

Adult↗