Biomedical subjects

P Gerlinger

Publications and source records attributed to P Gerlinger.

At least 19 recordsLinked to original sources

PubMed2003

[Towards a single embryo transfer: predictive value of uterine and embryological parameters in assisted reproduction outcome].

OBJECTIVE: The transfer of a single embryo would avoid obstetrical and neonatal complications due to multiple pregnancies. We studied the clinical value of both uterine and embryological parameters to define precise conditions allowing a single embryo transfer without decreasing pregnancy rates. PATIENTS AND METHODS: Endometrial parameters expressed by a uterine score together with biological criteria of 131 in vitro fertilization or intracytoplasmic sperm injection attempts were retrospectively analysed. RESULTS: Two hundred and sixty-two day-3 embryos were replaced through 131 transfers. Fifty-seven pregnancies were induced and 16 twins were obtained. The clinical pregnancy rate was 35.9% and the embryonic implantation rate 24.0%. After the transfer of two embryos, successful implantation was determined by the occurrence of top-quality embryos and simultaneously by a receptive endometrium. The uterine pulsatility index was significantly decreased for twin compared to singleton pregnancies. DISCUSSION AND CONCLUSION: This study confirms that a uterine score constitutes a powerful tool for evaluating the uterine receptivity. This parameter has to be taken into account as well as the embryonic quality, in order to optimise the success rate. Young patients with at least two top-quality embryos available, a high uterine score and a low pulsatility index, have a high risk of multiple births and are suitable for a single-embryo transfer.

Adult↗

PubMed2001

Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy.

We have developed a new allele-specific amplification method for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA; Werdnig-Hoffmann disease) from a single cell. This method is based on the detection of the deletion of exon 7 of the telomeric copy of the survival motor neurone (SMN(t)) gene. An oligonucleotide was designed to be specific to the SMN(t) nucleotidic sequence with exonic mismatch G (for SMN(t))-->A (for SMN(c)) at its 3' end. This test produces reliable PCR products in 95% of single lymphoblasts (85/88) tested as well as in 16/16 blastomeres from normal controls. Specificity analysis showed that we were able to detect homozygous deletion of the SMN(t) gene in 99% of single lymphoblasts (103/104) from a SMA patient. No contamination was detected in 68 blanks tested. Multiple cell and DNA dilution analysis revealed that the test is accurate and specific up to 100 pg DNA and should thus also be suitable for PGD at the blastocyst stage. This rapid procedure requires a single round of fluorescent PCR and no restriction digestion, while previously described single cell methods include nested PCR followed by restriction enzyme digestion. Two PGD cycles for SMA using this procedure were performed in our centre.

Adult↗

PubMed2001

[Twin birth after preimplantation diagnosis for cystic fibrosis].

OBJECTIVE: Cystic fibrosis is a common autosomal recessive disease most often caused by a deletion (delta F508) in the CFTR gene. It is the most common indication for preimplantaion genetic diagnosis which allows genetic analysis of embryos obtained after in vitro fertilization and transfer of unaffected embryos into the patient's uterus. PATIENTS AND METHODS: We report the first preimplantation genetic diagnosis performed in Strasbourg for a couple at risk of having a child affected by severe cystic fibrosis due to a homozygous delta F508 mutation. Three days after fertilisation, embryos obtained after intra-cytoplasmic testiculare sperm injection were biopsied and analysed. PCR amplification of the genomic fragment containing the delta F508 locus allowed detection of the delta F508 mutation and transfer only of the unaffected embryos. RESULTS: Three embryos were transferred after this preimplantation genetic diagnosis. A twin pregnancy was obtained and the babies born from this cycle are both exempt from the mutation. CONCLUSIONS: Preimplantation genetic diagnosis for the cystic fibrosis delta F508 mutation is now available in our centre. In this report, we could resolve both the problem of infertility and the risk of transmission of a severe form of cystic fibrosis. Preimplantation genetic diagnosis is also available for other mutations involved in cystic fibrosis and also for other genetic diseases.

Adult↗

PubMed2000

Do morphological anomalies reflect chromosomal aneuploidies?: case report.

In cases of severe teratozoospermia, the current morphological criteria used to assess chromosomal status is insufficient for the selection of spermatozoa for intracytoplasmic sperm injection (ICSI). Case histories are reported of four patients presenting 100% teratozoospermia, and the integrity of their individual chromosomal statuses is determined using a three-colour fluorescence in-situ hybridization (FISH) technique. Patient 1 presented shortened flagella syndrome, patient 2 globozoospermia, patient 3 spermatozoa with irregular acrosomes, and patient 4 macrocephalic spermatozoa with associated multiple flagella. Three-colour FISH analysis using chromosome X, Y and 1-specific probes showed that approximately 95% of the spermatozoa analysed from patients 1, 2 and 3 presented X,1 and Y,1 signals, X,Y ratios and aneuploidy/diploidy rates comparable with those observed in normal controls. In contrast, patient 4 showed a highly elevated Y to X sex ratio and a highly elevated aneuploidy/diploidy rate. Three-colour FISH analysis thus demonstrates an increased incidence of chromosomal abnormalities in association with macrocephalic spermatozoa. Moreover, the analysis shows that in patients affected with either globozoospermia, shortened flagella syndrome or a condition of abnormal acrosomal spermatozoa, no association exists between chromosomal status and phenotype. Since these patients display normal haploid, sex chromosome and aneuploidy status, ICSI can be conceivably offered as a treatment for their infertility.

Abortion, Spontaneous↗

PubMed2000

Zygote evaluation: an efficient tool for embryo selection.

One of the main problems concerning IVF units is the need to decrease the occurrence of multiple pregnancies in their practice without affecting the overall success rate. Different embryological parameters concerning every step of the early embryo development are known to have some predictive value for implantation potential. In this prospective study, a pronuclear scoring system was used to classify zygotes into six patterns from 0 to 5. Cleaved, day 3 embryos developed from pattern 0 zygotes, which was described as the normal pattern, were transferred when available. For each zygote pattern, the subsequent embryological development was analysed. Pattern 0 zygotes led to significantly more 'good quality' embryos with higher implantation potential than embryos developing from the other zygote patterns (P < 0.01). Embryo transfers including at least one pattern 0 resulted in significantly more pregnancies than transfers without any pattern 0 zygotes (39.3 versus 19.7%, P < 0. 01). No relationship between clinical parameters (age of female partner, infertility cause) and zygote pattern distribution was demonstrated.

Aging↗

PubMed1998

Preparing for preimplantation genetic diagnosis in France.

Preimplantation genetic diagnosis (PGD) allows the detection of genetic defects before implantation, thus circumventing the possible need for abortion. France's current legislation allows the practice of PGD under certain circumstances which include the prerequisite agreement of the French health authority. Unfortunately, to enact the pending 'bioethical law', voted in July 1994, a decree still needs to be published. So, for the moment, although we know that PGD should be authorized, its practice is currently impossible in France. In order to prepare for licensing, we are setting up the relevant technologies, by performing biopsy on mouse embryos and fluorescent in-situ hybridization (FISH) experiments on human lymphoblast cells. We briefly describe the French legal situation with regard to PGD and the work we have performed in this context to obtain the licensing to offer PGD to patients. After a period of preparation, 95.9% of biopsies were successful and up to 95.4% of the biopsied blastomeres were properly spread onto slides. Biopsied and control mouse embryos were reimplanted into pseudopregnant females and similar birth rates were obtained (34.4 and 30.9% respectively). In these experiments we noticed a birth delay of 12-24 h for the biopsied embryos compared with the controls. Furthermore, scanning electron microscopy of the biopsied embryos allowed assessment of the hole made by the Tyrode's acid. By intercrossing adults derived from biopsied embryos for two successive generations, it was shown that the biopsy did not generate defects affecting their reproductive ability. FISH experiments were performed using specific probes for chromosomes X, Y and 1 on nuclei spread by a conventional protocol or a Tween/HCl blastomere spreading protocol; in the latter case, slides with 1-5 cells were prepared. A similar percentage of correct X,Y,1,1 signal was obtained from all three types of spreading, varying from 85.5 to 89.9%.

Animals↗

PubMed1991

A case of human pregnancy after microinjection of capacitated sperm into the perivitelline space.

Microinjection of capacitated sperm into the perivitelline space of oocyte was offered to one couple with persistent infertility of mixed origin. The husband's semen was subnormal, whereas his wife had definitive tubal occlusion and polycystic ovaries. Four previous in vitro fertilization (IVF) attempts were performed but no fertilization was obtained. After superovulation, 13 oocytes were collected. Ten were submitted to microinjection and two were damaged during the procedure. One of the remaining eight had two pronuclei 18 hr after microinjection and progressed to a four-cell embryo after 48 hr. After reimplantation, a normal pregnancy was initiated, caryotype (46XX) was checked at 17 weeks. A normal and healthy girl has been delivered at term.

Adult↗

PubMed1991

The M-twist gene of Mus is expressed in subsets of mesodermal cells and is closely related to the Xenopus X-twi and the Drosophila twist genes.

The twist gene was characterized in Drosophila as being necessary at gastrulation for the establishment of the mesodermal germ layer. It codes for a nuclear DNA-binding protein that is probably a transcription factor. We have cloned and sequenced the M-twist gene of Mus musculus. The deduced proteins encoded by the Mus, Xenopus, and Drosophila twist cDNAs, respectively, show a high degree of similarity. Northern blot analyses and in situ hybridizations reveal that the 1.7-kb murine M-twist m-RNA is present at early stages, starting at 8 days post coitum, and is expressed the most at 9.5 days in the cephalic and branchial mesectoderm, in some derivatives of the mesodermal layer (sclerotoma and somatopleura), and in the limb buds.

Animals↗

PubMed1991

Precocious mammary gland development and milk protein synthesis in transgenic mice ubiquitously expressing human growth hormone.

A chimeric gene comprising the hydroxymethylglutaryl coenzyme-A reductase promoter and the human GH (hGH) genomic sequences was used to create transgenic mice expressing hGH in all tissues. In transgenic females, morphological development of the mammary gland and milk protein (WAP) expression commences at 3 weeks of age. At 8 weeks of age the mammary gland is morphologically and functionally comparable to that normally reached after 14-15 days of gestation. Precocious development correlated with local expression of hGH in mammary gland. Organ culture in the presence of different lactogenic hormones revealed that insulin and hydrocortisone are sufficient to maintain transcription of the WAP gene in transgenic mammary gland. In contrast, WAP transcription in normal gland required either hGH or PRL in addition to insulin and hydrocortisone. However, the effect of hGH on mammary differentiation does not appear to be solely mediated through an interaction with PRL receptors, since PRL, when added to cultured mammary tissues, did not elicit an equivalent response.

Animals↗

PubMed1991

H-ras induced transformation of mammary epithelium is favoured by increased oncogene expression or by inhibition of mammary regression.

The promoter of the mammary specific murine whey acidic protein gene was used to direct Ha-ras expression in different lines of transgenic mice. We found that this promoter contains a tissue specific enhancer which directed expression in both orientations albeit to different levels. We used this feature to generate low and high ras expressing transgenic lines. The reversed orientation led to a weak expression in lines 3 and 58 and to a tumor frequency of 2%. In contrast, 72% of mice from line 25 showing high ras expression developed mammary tumors. Nulliparity is one risk factor for human breast cancer, suggesting a protective effect of post-lactational mammary regression. In order to investigate the effect of post-lactational regression, the low tumor frequency lines were crossed with mice expressing ubiquitously the human growth hormone gene, which induces permanent development of the mammary epithelium. Indeed, mammary tumors were observed in 76% of double transgenic females. Thus, the tumorigenic potential of the ras oncogene in mammary cells in vivo correlates with the level of its expression and with the developmental history of the mammary gland. Transformation coincides with the escape of oncogene expression from the regulation of the Wap promoter and the extinction of endogenous Wap gene expression.

Animals↗

PubMed1990

Functional consequences of overexpressed Ia antigens in AK alpha/AK beta transgenic mice.

We report the creation and characterization of several transgenic mouse lines that carry genes coding for the Ak alpha or Ak beta MHC class II (or Ia) molecules. In all these lines, the transgenes are expressed at the RNA and protein level with correct tissue and cell type specificity. Crosses between certain of them yield progeny displaying very high surface levels of class II protein--roughly five times the normal amount--allowing us to evaluate the consequences of quantitative variation in Ia molecule density on the organization and function of the immune system. The effects appear rather limited: we detect subtle changes in thymic lymphocyte subpopulations, as well as an enhanced Ag presentation capacity in vitro. Yet, in vivo responses are largely unaffected, and Ia overexpression to such levels does not provoke lymphoproliferation, immunodeficiency, or autoimmunity.

Animals↗

PubMed1990

Heterologous protein expression by transimmortalized differentiated liver cell lines derived from transgenic mice (hepatomas/alpha 1 antitrypsin/ONC mouse).

A number of therapeutic plasma proteins are synthesized by human hepatocytes. Since many of these proteins undergo liver-specific post-translational modifications which are required for full biological activity, it may therefore be necessary to develop hepatocyte-based expression systems for their production. Using transgenic mice we have developed a transimmortalisation technique for the isolation of differentiated hepatic cell lines, already engineered to secrete human alpha 1 antitrypsin (alpha 1 AT), a plasma protein which is produced mainly in liver cells. This was achieved by co-expression of the mouse c-myc proto-oncogene and a genomic copy of the human alpha 1 AT gene, both under the control of the human alpha 1 AT promoter. Transgenic mice carrying this construct developed hepatomas producing human alpha 1 AT. Under defined culture conditions, cell lines secreting active alpha 1 AT were derived from these tumours. These cells maintain a differentiated hepatic phenotype and continue to secrete human alpha 1 AT for at least 40 generations.

Animals↗

PubMed1989

Analysis of the HLA-Cw3-specific cytotoxic T lymphocyte response of HLA-B7 X human beta 2m double transgenic mice.

The cytolytic responses of either normal (non transgenic), HLA-B7 (single transgenic) or HLA-B7 x human beta 2 microglobulin (double transgenic) DBA/2 mice induced by transfected HLA-Cw3 P815 (H-2d) mouse mastocytoma cells were compared, to evaluate whether the expression of an HLA class I molecule in responder mice would favor the emergence of HLA-specific, H-2-unrestricted CTL. Only 8 of 300 HLA-Cw3-specific CTL clones tested could selectively lyse HLA-Cw3-transfected cells in an H-2-unrestricted manner, all having been isolated after hyperimmunization of double transgenic mice. These clones also lysed HLA-Cw3+ human cells. Unexpectedly, the lysis of the human but not that of the murine HLA-Cw3 cells was inhibited by Ly-2,3-specific mAb. Despite significant expression of HLA-B7 class I molecules on transgenic lymphoid cells, including thymic cells, limiting dilution analysis and comparative study of TCR-alpha and -beta gene rearrangements of the eight isolated clones (which suggested that they all derived from the same CTL precursor) indicated that the frequency of HLA-Cw3-specific H-2 unrestricted cytotoxic T lymphocytes remained low (even in HLA-B7 x human beta 2-microglobulin double transgenic mice). This suggests that coexpression of HLA class I H and L chain in transgenic mice is not the only requirement for significant positive selection of HLA class I-restricted cytotoxic mouse T lymphocytes.

Animals↗

PubMed1989

Transgenic mice with I-A on islet cells are normoglycemic but immunologically intolerant.

Insulin-dependent diabetes mellitus (IDDM) is caused by a specific loss of the insulin-producing beta cells from pancreatic Langerhans islets. It has been proposed that aberrant expression of major histocompatibility complex (MHC) class II molecules on these cells could be a triggering factor for their autoimmune destruction. This proposal was tested in transgenic mice that express allogeneic or syngeneic class II molecules on the surface of islet cells at a level comparable with that normally found on resting B lymphocytes. These animals do not develop diabetes, nor is lymphocyte infiltration of the islets observed. This immunological inactivity does not result from tolerance to the "foreign" class II molecules.

Animals↗

PubMed1989

Restricted assembly of MHC class II molecules in transgenic mice.

Studies on cell lines transfected with MHC class II genes have revealed important limitations on the assembly of haplotype-mismatched A alpha:A beta complexes. These findings led to the speculation that pairing restrictions, if applied in a cell type-specific fashion, might be involved in various autoimmune phenomena. We have investigated pairing restrictions in vivo by analyzing transgenic mice that carry an Ak alpha chain, an Ak beta chain, or the Ak alpha:Ak beta complex on an H-2b or H-2s background. Our conclusion is that the assembly of haplotype-mismatched A alpha:A beta complexes is limited in vivo, and that this is equally true for all cell types examined, regardless of their role in the immune response.

Alleles↗

PubMed1989

Breast cancer protein PS2 synthesis in mammary gland of transgenic mice and secretion into milk.

PS2, a small estrogen-inducible secretory polypeptide with structural analogies to a growth factor, is produced by approximately 50% of human breast tumors. The function of PS2 is, however, unknown. To determine whether PS2 may play an autocrine role in the development of mammary tumors we constructed transgenic mice bearing fusion constructs designed to direct the expression of human PS2 in the lactating mammary gland under the control of the whey acidic protein (WAP) promoter. Mouse lines bearing the genomic PS2 gene under the control of the WAP promoter region (WAP-PS2-2) failed to express the transgene. However, mice harboring the fusion construct WAP-PS2-1, in which the PS2 coding sequence is inserted into the 5' untranslated region of the complete WAP gene, were observed to express the transgene. Expression was restricted to the secretory epithelium of the mammary gland during lactation, and PS2 protein was secreted into the milk. Nevertheless, no mammary gland dysplasia was observed, and PS2 expression had no discernable effect upon the physiology and/or development of the suckling young or the transgenic mother.

Animals↗

PubMed1988

Compartmentalization of MHC class II gene expression in transgenic mice.

A set of transgenic mouse lines carrying Ek alpha genes with promoter region deletions was created in an attempt to compartmentalize MHC class II gene expression. Fine immunohistological analyses established that one transgenic line is essentially devoid of E complex in the thymic cortex, another displays almost no E in the thymic medulla or on peripheral macrophages, and two lines display no E on greater than 98% of B cells. We have assayed these mice for immune function: E-dependent tolerance, antigen presentation, T cell priming, and antibody response. Certain of the findings are difficult to reconcile with currently popular hypotheses, e.g., tolerance induction to E molecules in the virtual absence of E complex in the thymic medulla and efficient antibody responses to E-restricted antigens when almost all B cells are E-.

Animals↗

PubMed1988

Targeted c-myc gene expression in mammary glands of transgenic mice induces mammary tumours with constitutive milk protein gene transcription.

We investigated the consequences of augmented c-myc gene expression in the mammary gland of transgenic mice. For this purpose we directed the expression of a mouse c-myc transgene to the differentiating mammary epithelial cells by subjecting the protein coding region to the 5' regulatory sequences of the murine whey acidic protein gene (Wap). Analogous to the expression pattern of the endogenous Wap gene, the Wap-myc transgene is abundantly expressed in the mammary gland during lactation. The tissue-specific and hormone-dependent expression of the Wap-myc transgene results in an 80% incidence of mammary adenocarcinomas. As early as two months after the onset of Wap-myc expression, tumours occur in the mammary glands of the transgenic animals. The tumours express not only the Wap-myc transgene, but also the endogenous Wap and beta casein genes. The expression of the milk protein genes becomes independent of the lactogenic hormonal stimuli and persists even in transplanted nude mouse tumours.

Adenocarcinoma↗