Biomedical subjects

J Lornage

Publications and source records attributed to J Lornage.

At least 19 recordsLinked to original sources

PubMed2006

[Ovarian tissue vitrification: Cortex and whole ovary in sheep].

OBJECTIVE: The aim of this study was to evaluate a cryopreservation technique by vitrification of cortex or whole ovaries in sheep, using two cryoprotectant solutions: VS1 and VS4 and to study their physical properties to avoid ice crystallisation by vitrification of whole sheep ovaries permeated with a cryoprotectant solution. ANIMALS AND METHODS: From 6-month-old ewes, whole sheep ovaries with their vascular pedicles were collected at the slaughterhouse or at the veterinary school and prepared for cryoprotectant toxicity tests and freezing procedure. Follicle viability was measured by trypan blue test and histological examination of ovary. The hemi-ovarian cortex was stored in liquid nitrogen. Four to six weeks after the first laparotomy, the controlateral ovary was removed and the vitrified-warmed hemi-ovary was sutured. Thermal properties of a cryoprotectant solution called VS4 (critical cooling rates [Vccr], vitreous transition temperature [Tg], end of melting temperature [Tm]) were measured by differential scanning calorimetry. RESULTS: No significant difference in follicle viability or normal follicle rates was observed between ovarian cortex exposed or non-exposed to cryoprotectant solutions. Nor was any significant difference observed before and after vitrification. Three pregnancies occurred, from which four lambs were born after autografts of vitrified ovarian cortex. With whole ovary, the decrease in the number of normal follicles was lower when frozen-thawed ovaries were treated with VS4 (P = 0.04). There were less nuclear anomalies (P = 0.02). The Vccr of VS4 has been estimated to be 14.3+/-1.1 degrees C/min and Tg was -125.0+/-0.2 degrees C. Because the penetration of cryoprotectants was very low, Vccr was very high and the cooling speed did not allow cortex to vitrify. DISCUSSION AND CONCLUSIONS: Cryopreservation of cortex or whole ovary by vitrification seems a promising technique in reproductive medicine. The best histologic results were obtained with the VS4 cryoprotectant when whole ovary was vitrified.

Animals↗

PubMed2006

[In vitro maturation: the French experience].

In vitro oocyte maturation (IVM) allows the use of immature oocytes in IVF. IVM does not require ovarian stimulation and therefore can be offered to patients at risk of ovarian hyperstimulation syndrome. IVM can be offered as an alternative to conventional IVF in women with PCOS (polycystic ovarian syndrome). However, cleavage, implantation, and pregnancy rates were lower than those obtained with conventional IVF. The present study describes the results of IVM in French IVF departments.

Cells, Cultured↗

PubMed2005

Influence of global sperm DNA methylation on IVF results.

BACKGROUND: In cases of male infertility, routine analysis for sperm characteristics is a poor predictive factor for the segmentation rate and embryo development in assisted reproductive technologies. It is assumed that epigenetic factors could have an influence on the embryo's quality. The aim of this work was to determine the relationship between sperm DNA methylation level and fertilization and pregnancy rates according to the assisted reproduction technique performed. METHODS: A prospective study was undertaken. Ejaculates were obtained from men (n = 63) undergoing an assisted reproduction procedure. 5-Methylcytosine was immunostained with a polyclonal antibody and revealed by fluorescein isothiocyanate. The DNA methylation level was then quantified by flow cytometry. RESULTS: Sixty-three conventional IVF cycles were performed, 760 oocytes were retrieved, an average of 8.1 +/- 4.8 embryos was obtained, and 2.4 embryos were transferred. Neither the fertilization rate nor the rate of good quality embryos was correlated with the DNA methylation level (r = -0.1 and r = -0.08 respectively; not significant). When sperm DNA methylation was >555 arbitrary units, the pregnancy rate was 33.3% compared with 8.3% in the lower (<555) group (P<0.05). CONCLUSION: DNA methylation level in human sperm could represent a new approach to study the ability of sperm to lead to pregnancy in an assisted reproduction procedure, especially when sperm samples with normal characteristics are used.

5-Methylcytosine↗

PubMed2005

[Follicular morphology after ovarian cortex cryopreservation in the rabbit doe(Oryctolagus cuniculus)].

OBJECTIVE: To compare the effects of two cryoprotective agents (DMSO and 1,2-PROH) used at two concentrations (1,5 and 2 M) on the morphology of small ovarian cortex follicles in doe. MATERIALS AND METHODS: Ovarian cortexes (n=40) were frozen in TCM199+10% FCS medium added to 1.5 or 2 M of DMSO or 1,2-PROH. Two controls were realized (fresh and frozen without cryoprotectant). The equilibration in cryoprotective solutions before freezing, and the elimination of the cryoprotective agents after thawing, was performed step by step. The effects induced by cryopreservation were evaluated by histological examination. RESULTS: Fresh ovarian tissue showed 68.6% of intact follicles. After freezing, only 1.5 M of 1,2-PROH preserved 48.0% of normal follicles, with no significant difference compared to the fresh control. The proportion of follicles without morphological defect observed after cryopreservation with DMSO was significantly reduced (respectively 28.8 and 34.8% for 1,5 and 2 M of DMSO). DISCUSSION AND CONCLUSIONS: Our results suggest that 1,2-PROH is a more effective cryoprotectant than DMSO, for the cryopreservation of doe ovarian cortex. These results differ from those that were obtained for other species, credibly because of a higher fragility of the ovarian tissue of the doe. Nevertheless, this species is an interesting animal model which allows rapid results after cryopreserved ovarian tissue graft.

Animals↗

PubMed2003

[Biological aspects of endometriosis in vitro fertilization].

Rinsing cumulus oophorus after oocyte retrieval is routinely performed in our department in Lyon for in vitro fertilization procedures when ovarian endometriosis is suspected. The aim of this procedure is to remove factors which might alter oocyte and embryo quality and fertilization and pregnancy rates. These factors, such as Tumor Necrosis Factor alpha (TNFalpha) and reactive oxygen species are known to be surexpressed in endometriosis. Their effect is not exclusively on the development of the oocyte and embryo but also on fertilization and implantation. It has been demonstrated that peritoneal and follicular fluid contains some factors that interfere with effective gamete interaction, as it has been determined by an in vitro animal assay. Peritoneal fluid from patients with mild endometriosis has an increased activity of TNFalpha, interleukin-1 (IL-1), antibodies and reactive oxygen species. This observation might be of importance in explaining subfertility associated with early stages of endometriosis. Cumulus-oophorus rinsing after oocyte retrieval might help to remove these deleterious factors. IVF outcomes in women affecting by endometriosis seem to be similar after cumulus-oophorus rinsing, as compared to those of patients undergoing IVF for tubal-factor infertility. This rinsing procedure could also be usefull in women affected by unexplained infertility: peritoneal and follicular fluid of these women contains increased levels of TNFalpha and reactive oxygen species, like women affected by endometriosis.

Endometriosis↗

PubMed2002

Pregnancy after safe IVF with hepatitis C virus RNA-positive sperm.

In France, assisted reproductive technology (ART) for hepatitis C virus (HCV)-infected patients is now subject to strict control after the publication of recent guidelines. Infertile serodiscordant couples (HCV-viraemic men and their seronegative female partners) require special care to carried out in designated 'viral risk' laboratories. Twelve sequential semen samples taken from an HCV chronically infected patient were analysed within 22 months. HCV RNA was detected in all the seminal plasma sampled before antiviral treatment with relatively high viral loads, and in two of the corresponding fractions of motile sperm obtained after a gradient selection, suggesting that a contamination risk by HCV through ART cannot be excluded. When the selection of sperm on a discontinuous gradient was followed by an additional swim-up step, HCV RNA was never detected in the motile sperm suspension that was frozen in highly secure straws. IVF was performed using cryopreserved sperm that tested negative for HCV RNA, resulting in a pregnancy. One month after embryo transfer, testing for HCV RNA and antibodies in the woman gave negative results.

Adult↗

PubMed2001

Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols.

OBJECTIVE: To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex. DESIGN: Small follicles (<60 microm in diameter) were isolated enzymatically for viability testing. Dead and live follicles were identified by using trypan blue staining, and follicle morphology was examined histologically. SETTING: Centre hospitalo-universitaire de Biologie de la Reproduction, Hôpital Edouard Herriot, Lyon, France. ANIMAL(S): Lambs 5 to 6 months of age. INTERVENTION(S): Two-millimeter slices of hemiovarian cortex were prepared for cryoprotectant toxicity tests and freezing procedures. MAIN OUTCOME MEASURE(S): Follicular mortality and histologic structure. RESULT(S): For freezing procedures, the concentration of cryoprotectant was increased to 2 M on the basis of results of cryoprotectant toxicity tests in fresh tissues. Follicular mortality rates were 4.6% with of 2 M dimethyl sulfoxide (DMSO) and 3.8% with 2 M of propylene glycol (PROH). After freezing with semiautomatic seeding, follicular mortality rates were 8.4% (2 M of DMSO) and 12.4% (2 M of PROH). Tissue morphology was well preserved with 1.5 M of DMSO or PROH. With 1.5 M DMSO, results of the slow cooling protocol (2 degrees C/min) without seeding and the standard very slow cooling protocol (0.3 degrees C/min) were similar. CONCLUSION(S): Optimal survival of primordial follicles in the sheep was obtained by using a slow cooling protocol with semiautomatic seeding at 2 M of DMSO.

Animals↗

PubMed2001

Comparison of two blastocyst culture systems: coculture on Vero cells and sequential media.

OBJECTIVE: To compare two blastocyst culture systems: culture on Vero cells and sequential media in the same time period. DESIGN: Retrospective study. SETTING: Institutional practice assisted reproductive technology center. PATIENT(S): Ninety-nine selected patients undergoing IVF with blastocyst culture. INTERVENTION(S): In vitro fertilization and transfer of at least one expanded blastocyst after culture using either Vero cells or sequential media (S(2) medium). MAIN OUTCOME MEASURE(S): Blastocyst, pregnancy, and implantation rates. RESULT(S): The blastocyst formation rate (41.6% vs. 31.6%) is significantly increased with sequential media compared with the coculture system, but there is no statistically significant difference in the pregnancy and implantation rates (39.5% vs. 38.6% and 23.7% vs. 15.1%, respectively). CONCLUSION(S): Blastocyst culture on sequential media offers a better dynamic of embryo development (22% vs. 13.6% blastocyst at day 5). There was no statistically significant difference between the two groups in the take-home baby rate (18 vs. 12).

Adult↗

PubMed2000

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.

Blotting, Western↗

PubMed2000

Intra- and inter-individual variability in human sperm concentration, motility and vitality assessment during a workshop involving ten laboratories.

The aim of the present study was to assess variability in the evaluation of human sperm concentration, motility and vitality. Technicians and biologists from 10 teams involved in multicentre studies on semen quality attended the same laboratory, each team using its own methods and equipment to analyse the same semen samples. Inter-individual variability was assessed from 17 fresh semen samples of varying quality. Intra-individual variability was assessed from pools of frozen samples for sperm concentration and motility and stained smears for vitality with three blind evaluations by sample and smear. The mean inter-individual coefficients of variation were 22.9, 21.8 and 17.5% for sperm concentration, motility and vitality respectively. There was no statistical difference among participants for sperm concentration assessment, but significant differences for both motility and vitality (both P: < 0.05). The mean intra-individual coefficients of variation were 15.8, 26.2 and 13.1% for sperm concentration, motility and vitality respectively, with marked differences between expert and novice participants: concentration 9.8% versus 28.0%; motility 22.8% versus 33.0%; and vitality 10.0% versus 19.3%. The present data confirm the need for external quality control schemes for diagnostic purposes, and indicate their utmost importance in multicentre studies on semen quality.

Cell Survival↗

PubMed2000

Transmission risk of hepatitis C virus in assisted reproductive techniques.

Medical assistance for procreation in a couple where one or both parents has hepatitis C viral infection (HCV) raises the issue of the transmission of the infection to the baby and/or of possible contamination of both the technicians and the gametes or embryos from virus-free parents in the laboratory. It becomes essential to assess transmission risk in assisted reproductive techniques in order to define clearly the management of couples according to their viral status. To define the HCV transmissibility risk in assisted reproduction related to the presence of virus in semen from infected infertile men, HCV RNA detection was performed in sera, and semen and sperm fractions obtained after Percoll gradient centrifugation. HCV RNA was detected in 5% (2/39) of the semen samples tested: in the raw semen, in the seminal fluid and in the cell pellet but never after Percoll selection. According to these results, we suggest a strategy for HCV-infected infertile men who need assistance for procreation.

Adult↗

PubMed2000

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.

Cysteine Endopeptidases↗

PubMed1999

Restoration of ovarian steroid secretion and histologic assessment after freezing, thawing, and autograft of a hemi-ovary in sheep.

OBJECTIVE: To evaluate the effects of freezing, thawing, and autograft of a hemi-ovary on steroid secretion, endometrial maturation, and ovarian histology in ewes. DESIGN: Experimental animal study. SETTING: Laboratoire de Zootechnie, Ecole Nationale Vétérinaire, Marcy l'Etoile, France. ANIMAL(S): Six lambs aged 6 months to 1 year old. INTERVENTION(S): Hemi-ovaries were prepared and frozen from the right ovary of six lambs and autografted 4 weeks later to the contralateral ovarian hilus. The autografts and the uterus were recovered 1 year later. Blood tests were performed each week to measure P concentration. MAIN OUTCOME MEASURE(S): Number of primordial follicles; levels of plasma P. RESULT(S): Histologic examination of ovarian slices after freezing showed no destruction of primordial, primary, secondary, or cavitary follicles. The frozen ovarian autograft showed good recovery of the macroscopic and microscopic ovarian structure. After autografting, histologic examination revealed primordial to cavitary follicles. Secretion of P started to rise 4 weeks after the autograft. Histologic analysis of the endometrium showed numerous glands, vessels, and mucous secretion. CONCLUSION(S): Frozen ovarian autografts achieved P secretion and endometrial maturity.

Animals↗

PubMed1999

Screening for Chlamydia trachomatis and Ureaplasma urealyticum infection in semen from asymptomatic male partners of infertile couples prior to in vitro fertilization.

In a prospective study, the prevalence of infection with Chlamydia trachomatis and Ureaplasma urealyticum was evaluated in the semen of 92 asymptomatic male partners of infertile couples using polymerase chain reaction and culture, respectively. The results were compared with the detection of serologically specific antibodies. U. urealyticum and C. trachomatis were detected in 12 (13%) and 10 (10.8%) of the tested ejaculates, respectively. One mixed infection was detected. No correlation was found between detection of the pathogens in ejaculates and the presence of specific antibodies in serum. This study therefore confirms the limited diagnostic value of serological analysis to ascertain infection with C. trachomatis or U. urealyticum. The high frequency of detection of these pathogens among asymptomatic male partners of infertile couples emphasizes their potential role in the impairment of male fertility, and the need for sensitive and specific detection methods to prevent infection of the early embryo when using new reproductive techniques such as zona pellucida hatching or intracytoplasmic microinjection.

Adult↗

PubMed1998

Freezing, thawing, and autograft of ovarian fragments in sheep: preliminary experiments and histologic assessment.

OBJECTIVE: To evaluate the effects of freezing and thawing on the histologic changes in ovarian fragments from sheep and to determine the feasibility of ovarian autografts. DESIGN: Histologic evaluation of follicles that survived after freezing at -196 degrees C for 2 weeks. Histologic evaluation of ovarian fragments 6 months after the autografts. SETTING: Laboratoire de Zootechnie, Ecole National Véterinaire, Marcy l'Etoile, France. ANIMAL(S): Six ewes aged 6 months to 1 year. INTERVENTION(S): Cortical fragments were prepared from the right ovary of 6 lambs and were grafted immediately to the contralateral ovarian hilus or were cooled slowly to -196 degrees C in medium containing dimethyl sulfoxide for 2 weeks. The autografts were recovered 6 months later. RESULT(S): Histologic examination of ovarian slices after freezing showed no destruction of primordial, primary, secondary, or cavitary follicles. The ovarian autograft showed good recovery of the macroscopic and microscopic ovarian structure. After autografting, histologic examination revealed primordial to cavitary follicles. CONCLUSION(S): Freezing of ovarian fragments is possible without damaging the follicles. Ovarian autografts showed good recovery of ovarian structure.

Animals↗

PubMed1997

Detection of cytomegalovirus in semen from a population of men seeking infertility evaluation.

OBJECTIVE: To determine the incidence of cytomegalovirus in the ejaculates of infertile men who were seropositive for IgG antibodies to cytomegalovirus. DESIGN: Prospective study. PATIENT(S): We tested cytomegalovirus infection in the semen of men participating in an IVF-ET program. MAIN OUTCOME MEASURE(S): IgG and IgM antibodies to cytomegalovirus were measured in sera. We used polymerase chain reaction (PCR) and cell culture to look for both cytomegalovirus DNA and infectious virus in the semen of 70 men with cytomegalovirus-specific antibodies detected in sera. RESULT(S): Of the infertile couples, 13.5% exhibited "mismatching" serology (i.e., detection of IgG antibodies to cytomegalovirus in male serum only and not in female serum) and constituted a potential risk for cytomegalovirus transmission. Cytomegalovrius was identified in the semen of two patients who were positive for IgG antibodies to cytomegalovirus. Cytomegalovirus DNA also was detected in one positive sample after centrifugation through a three-layer Percoll gradient. CONCLUSION(S): Human cytomegalovirus was present in the semen from a population of infertile men. Rapid detection can be achieved by molecular techniques such as PCR combined with a hybridization assay. Even though cytomegalovirus was infrequently detected in semen, these data must be considered in determining the risk of transmission and developmental anomalies in infected fetuses.

Adult↗

PubMed1996

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.

Acrosome↗

PubMed1996

Uterine rupture in first or second trimester of pregnancy after in-vitro fertilization and embryo transfer.

We report five cases of early rupture of cornual pregnancy with history of previous salpingectomy and cornual resection following in-vitro fertilization (IVF) and embryo transfer. We discuss the predisposing factors, diagnostic and therapeutic modalities in these patients. A high index of suspicion is required for an early diagnosis. It is imperative that the physicians who care for the patients be fully aware of the possibility of such a complication in a high risk population; therefore, appropriate counselling and close follow-up might help to avoid such obstetrical catastrophes, by termination of pregnancy, either surgically or medically.

Adult↗