[Indications of primary cesarean deliveries in a regional teaching hospital and reasonable strategies for reducing them]. / Indications des premières césariennes dans un centre hospitalo-universitaire régional et stratégies raisonnables pour les diminuer.

OBJECTIVES: To characterize the indications of primary cesarean sections and discuss the various possibilities to reduce them. MATERIALS AND METHODS: Retrospective study, carried out over a period of 1 year in a university hospital having a level 3 perinatal activity, including the 499 primary cesarean sections of 2013. Two groups were defined by parity: nulliparous patients (group 1) and multiparous patients who had never previously been delivered by cesarean section (group 2). We have assessed the indication of every primary cesarean section and health status of newborns in each group. RESULTS: Groups 1 and 2 respectively included 369 and 130 patients. The cesarean section rate in 2013 was 24.7% with a primary cesarean section rate of 17%. Seventy-four percent of the primary caesarean deliveries were performed on nulliparous women and 26% on multiparous (P<0.001). Sixty-three percent of the primary caesarean deliveries were performed on nulliparous women with a singleton fetus in cephalic presentation. The most common indications for primary cesarean delivery were non-reassuring fetal heart rate tracing (47.1%), failure to progress (24.8%) for which nulliparous women were more involved (29% vs. 13%, P<0.001) and fetal malpresentation (9.6%). CONCLUSION: Further analysis of fetal heart rate during labor, a larger use of second line means to evaluate the fetal status during labor, using 6cm as the cut off for active labor, and encouraging vaginal operative delivery constitute the best way to decrease the primary cesarean section rate.

Variable content of Fel d 1 variants in house dust and cat extracts may have an impact on allergen measurement.

BACKGROUND: The major cat allergen, Fel d 1, is a tetrameric glycoprotein composed of 2 heterodimers. Polymorphisms in this allergen are well documented. Recent work shows that Fel d 1 samples can contain core fragments of variable immunoreactivity. OBJECTIVES: Our objective was to compare Fel d 1 polymorphism in cat extracts and house dust, which is used as an indicator of allergen exposure and to understand how the combination of individual Fel d 1 variants can affect cat allergen measurement. METHODS: Natural Fel d 1 allergens were water-extracted from house dust and from the chest area and anal sacs of a cat. Recombinant Fel d 1 was provided commercially. The samples were analyzed by immunoblotting; variants were isolated using gel electrophoresis and tested using enzyme-linked immunosorbent assay. RESULTS: Four Fel d 1 variants of 40, 30, 19-21, and 14-16 kDa were consistently identified in Fel d 1 samples. Fel d 1 patterns found in house dust and the chest area wash were similar. Dimers were shown to be the major variant, while intact or truncated tetramers and core fragments were found in variable amounts. Intact and truncated dimers of Fel d 1 displayed similar antibody binding. Conversely, the intact tetramer-but not the core tetramer-was found to bind twice the antibody amount as the dimers and core fragments. CONCLUSIONS: Despite a common pattern of Fel d 1 variants in cat extracts and house dust, variations in the tetramer-to-dimer ratio among samples may introduce major discordances in cat allergen measurements using immunoassays. Our findings indicate the need for further harmonization of allergen immunoassays.

Exploring the acceptor substrate recognition of the human beta-galactoside alpha 2,6-sialyltransferase.

Human beta1,4-galactoside alpha2,6-sialyltransferase I (ST6GalI) recognition of glycoprotein acceptors has been investigated using various soluble forms of the enzyme deleted to a variable extent in the N-terminal half of the polypeptide. Full-length and truncated forms of the enzyme have been investigated with respect to their specificity for a variety of desialylated glycoproteins of known complex glycans as well as related proteins with different carbohydrate chains. Differences in transfer efficiency have been observed between membrane and soluble enzymatic forms, indicating that deletion of the transmembrane fragment induces loss of acceptor preference. No difference in substrate recognition could be observed when soluble enzymes of similar peptide sequence were produced in yeast or mammalian cells, confirming that removal of the membrane anchor and heterologous expression do not alter enzyme folding and activity. When tested on free oligosaccharides, soluble ST6GalI displayed full ability to sialylate free N-glycans as well as various N-acetyllactosaminyl substrates. Progressive truncation of the N terminus demonstrated that the catalytic domain can proceed with sialic acid transfer with increased efficiency until 80 amino acids are deleted. Fusion of the ST6GalI catalytic domain to the N-terminal half of an unrelated transferase (core 2 beta1,6-N-acetylglucosaminyltransferase) further showed that a chimeric form of broad acceptor specificity and high activity could also be engineered in vivo. These findings therefore delineate a peptide region of approximately 50 amino acids within the ST6GalI stem region that governs both the preference for glycoprotein acceptors and catalytic activity, thereby suggesting that it may exert a steric control on the catalytic domain.

Remodeling of glycoprotein and carbohydrate antigens.

Glycosylation is the most extensive of all post-translational modifications in proteins. It has important functions in their secretion, antigenicity and metabolic clearance through structural polymorphism. In recent years, advances in recombinant DNA technology allowed the production of recombinant therapeutic proteins, among which glycosylated proteins displayed differences compared to their native counterparts, including antigenic carbohydrates. In this review, we discuss the potential use of cloned glycosyltransferases in remodeling recombinant glycoprotein antigens as well as in synthesizing tumor-associated carbohydrate antigens.

Glycosylation is the structural basis for changes in polymorphism and immunoreactivity of pituitary glycoprotein hormones.

Glycoprotein hormones have long been known to display extensive polymorphism and changes in bioactivity according to the endocrine status of the patient. Structural analysis has shown that pituitary gonadotropins (lutropin and follitropin) and thyrotropin are synthesized and secreted as a panel of isoforms which differ in glycosylation, bioactivity and circulatory half-life. Ultrasensitive immunoassays could reveal that glycosylation of plasma hormones is structurally different from the pituitary stock so that the ratio of circulating glycoforms may vary according to the physiopathology of the pituitary axis. However, contradictory results between immunoassays have been often reported, suggesting that some plasma forms can escape recognition by monoclonal antibodies which have been raised to the pituitary or urinary antigen. When hormone levels do not correlate with clinical features, one can also suspect that inactive or hyperactive forms are being measured. At the molecular level, very limited information has been gained toward the expression of hormone epitopes as a function of carbohydrate structure. To address this issue, we have compared the recognition of pituitary and recombinant human thyrotropin by various polyclonal and monoclonal antibodies before and after neuraminidase treatment. Both, pituitary and recombinant thyrotropin bound to anti-alpha and anti-beta antibodies, demonstrating thereby that recombinant thyrotropin can be used to calibrate immunoassays. While removal of sialic acid did not alter the recognition of the recombinant hormone in various immunoassays, this treatment specifically abolished the binding of pituitary thyrotropin to anti-beta monoclonal antibodies. These findings show that immunoreactivity of circulating hormone glycoforms, which are often more sialylated than their pituitary counterparts, may very well account for variation depending on the antibodies used in the immunoassays.

Biological and immunochemical characterization of recombinant human thyrotrophin.

Recombinant human thyroid-stimulating hormone (recTSH) has recently been engineered to detect metastatic lesions in patients operated on for thyroid cancer. In this report, we have compared the microheterogeneity, carbohydrate (CHO) content, mitogenic potency and immunoreactivity of the biotechnology product to those of human TSH of pituitary origin (pitTSH). Compositional analysis revealed that recombinant (rec) TSH produced in Chinese hamster ovary cells was overglycosylated compared with the native hormone (21 and 14%, respectively) with a higher amount of sialic acid and lack of N-acetylgalactosamine. Electrofocusing followed by immunoblotting resolved recTSH into six glycoforms with pIs ranging from 6.0 to 8.6, which were converted to a major species of pI 8.9 by sialidase treatment. pitTSH contained five main isoforms of pI 6.5-8.2 distinct from those of recTSH and partially resistant to sialidase. Binding activity of both human TSHs to porcine thyroid membrane receptors was found to be similar, but recTSH appeared to be 20% active compared to pitTSH in eliciting cAMP production and cell growth in rat FRTL-5 cells. Immunoreactivity of the recombinant hormone was investigated using polyclonal and monoclonal antibodies raised against the native hormone or synthetic peptide sequences of its subunits. While rec- and pitTSH were recognized to a similar extent by anti-protein antibodies, they exhibited a different binding pattern to antipeptide antibodies. Serial dilution of anti-alpha 1-25, anti-alpha 26-51, anti-beta 96-112 antisera bound recTSH to a greater extent than pitTSH, while anti-beta 31-51 and anti-beta 53-76 displayed similar recognition toward both preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual activity of human pituitary thyrotrophin isoforms on thyroid cell growth.

Alkaline (pI 8.6-7.5) and neutral (pI 7.0-6.0) isoforms of human TSH have been isolated from a highly purified intrapituitary preparation by isoelectric focusing and compared for their respective actions on thyroid cell proliferation. Both TSH isoforms displayed the same ability to bind to porcine thyroid membranes as the original hormone preparation, indicating a similar recognition at the receptor sites. Alkaline forms showed a higher potency in inducing either cyclic AMP (cAMP) production or [3H]thymidine incorporation in FRTL-5 cells (half-maximal effective doses (ED50 values) = 0.25 and 0.29 nM respectively) compared with their neutral counterparts (ED50 values = 0.66 and 0.70 nM respectively). Increasing the concentration of alkaline forms in the presence of a half-maximal concentration of neutral TSH resulted in a profound inhibition of cell growth without a significant change in cAMP. Conversely, increasing the amount of neutral forms in the presence of a half-maximal dose of alkaline TSH resulted in an additive response for cAMP production but not in cell proliferation. To assess whether glycosylation might be responsible for the variation in hormone action, both alkaline and neutral TSH isoforms were tested for recognition of their carbohydrate chains by concanavalin A (Con A) and ricin. No major difference was found in binding to Con A, indicating that the contribution of carbohydrates to changes in hormone pI was not related to core branching. Very few galactose residues were accessible in either hormone fraction since little binding to ricin was observed. Isoelectric focusing of TSH forms before and after neuraminidase treatment revealed that neutral forms had a higher sialic acid content than alkaline TSH. In conclusion, the current findings show that TSH isoforms differentially affect cAMP production and cell growth. TSH fractions with a high sialic acid content and a low mitogenic activity behave as antagonists to the more active forms for cell proliferation. It is suggested that physiological control of TSH action at the thyroid gland may reside in the respective amounts of various TSH forms which, once bound to their receptor, can induce variable activation of post-receptor events while controlling cell proliferation.

Glycosylation of human prolactin regulates hormone bioactivity and metabolic clearance.

To analyze the role of individual glycosylation pattern on PRL biopotency, monomeric prolactin (PRL), secreted by human prolactinoma cells in culture, was isolated by gel filtration and separated by affinity chromatography on Concanavalin A-Sepharose or Lentil-Agarose. These lectins allowed the isolation of PRL glycoforms containing either biantennary, mannose-rich or fucosylated complex carbohydrate structures, respectively. Endoglycosidase treatment and carbohydrate content of PRL was found to be consistent with N-linked oligosaccharides of mannose-rich structure and complex units terminated in sialic acid. Mannose-rich PRL and PRL with biantennary oligosaccharides promoted cell growth of rat lymphoma cells to a diminished extent compared to non-glycosylated PRL (NG-PRL), indicating that the two major types of carbohydrate structure are able to decrease the intrinsic bioactivity of PRL. Metabolic clearance of the various forms of PRL in rats was also found to be highly dependent upon hormone glycosylation. The various glycosylated forms (G-PRLs) proved to be totally eliminated from the circulation within 60 min, faster than NG-PRL 10% of which was still present at that time. Mannose-rich or biantennary G-PRLs were differently cleared in a biphasial fashion with a similar rapid phase of about 2 min followed by distinct slow phases of 12 and 27 min, respectively. The presence of fucose did not alter this distribution. In contrast, NG-PRL was eliminated with a half-time of approximately 5 min, followed by a very slow disappearance over several h. It thus appeared that glycosylation increased the metabolic clearance rate of PRL from 0.13 +/- 0.07 ml/min for NG-PRL to 0.47 +/- 0.12 ml/min for PRL with biantennary carbohydrate chains and 0.8 +/- 0.2 ml/min for the hormone with mannose-rich oligosaccharides. The distribution of PRL to target and elimination organs was also found to be different according to the carbohydrate structure present in the hormone. NG-PRL and mannose-rich G-PRL showed higher incorporation in liver than biantennary G-PRL which was preferentially eliminated by the kidney. Altogether, the current data show that addition of oligosaccharides to PRL as well as carbohydrate structure contribute to modulate both the duration of the hormone in the blood and its distribution to different organs. It is proposed that glycosylation may selectively down-regulate PRL action at individual target tissues.

Variable carbohydrate structures of circulating thyrotropin as studied by lectin affinity chromatography in different clinical conditions.

Carbohydrate structures of intrapituitary and circulating TSH were studied by Concanavalin-A (Con A) and ricin lectin chromatography under different clinical conditions. Con A permits the separation of molecules differing in the extent of their carbohydrate branching, whereas ricin gives an estimation of the degree of their sialylation. Intrapituitary TSH was more retained on Con A and less sialylated than circulating hormone, suggesting that carbohydrate chains of intrapituitary molecules are less mature than those present in the circulation. A greater proportion of TSH firmly bound to Con A, compared to control values, was found in sera from fetuses and patients with uremia, TSH-secreting adenomas, and central hypothyroidism. In primary hypothyroid patients, TSH binding to Con A was similar to that found in controls, but a greater percentage of sialylated forms was seen. In central hypothyroidism patients, TSH released in response to TRH was less sialylated. Interestingly, no sialylated TSH was found in normal fetuses. In conclusion, the present data show that both TSH carbohydrate branching and sialylation may vary in different clinical conditions. As some of the above clinical conditions are known to be accompanied by variations in the bioactivity of circulating TSH, the finding of changes in TSH carbohydrate structures further supports the view that glycosylation modulates the expression of TSH biological activity.

Concanavalin A affinity chromatography of human serum gonadotropins: evidence for changes of carbohydrate structure in different clinical conditions.

We have studied the carbohydrate of circulating human gonadotropins (FSH and LH) in different clinical conditions using Concanavalin A (Con A) affinity chromatography. This technique permits separation of molecules differing in the extent of carbohydrate branching. The proportion of molecules that does not bind to Con A was greater in circulating FSH than in LH, reflecting a higher content of multiantennary and/or bisected biantennary complex carbohydrate structures in serum FSH. No significant difference in gonadotropin binding pattern to Con A was found between normal controls and patients with chronic uremia or gonadotropin-secreting pituitary adenomas. On the contrary, sera from postmenopausal women and fetuses contained a greater proportion of FSH and LH that bound to Con A, indicating a shift from multiantennary and/or bisecting structures to hybrid and/or high mannose forms, i.e. to the secretion of less mature forms. International Reference Preparations, derived from pituitary extracts, were more retained on Con A than circulating hormones, suggesting that carbohydrate chains of the intrapituitary hormone stock are less mature than those present in the circulation. Less mature forms were also found in FSH, but not in LH, from normal controls after GnRH injection. Finally, a higher proportion of unbound forms, i.e. complex carbohydrate chains, was found in healthy subjects presenting with an immunologically anomalous variant of LH. In conclusion, the current data show that the hormonal status of the individual may differently affect carbohydrate branching of gonadotropins. Alteration in glycosylation is likely to be involved in masking at least one epitope specific for intact LH dimer, thus indicating that it may modulate the tertiary structure of glycoprotein hormones.

Polymorphism of thyrotropin and alpha subunit in human pituitary adenomas.

To understand better why patients with TSH-secreting pituitary tumors exhibit variable degree of hyperthyroidism, we analyzed the various isoforms of TSH and alpha-subunit secreted by 4 TSH-secreting adenomas in primary culture. All patients had macrodenomas clinically associated with hyperthyroidism with normal to elevated TSH plasma levels. The in vivo molar alpha/TSH ratio ranged from 18.4 to 3.8. The hormone material secreted over 4 to 48 h in culture was separated by gel isoelectrofocusing, eluted and estimated by immunoassays. The release of free alpha-subunit was noticeably different among adenomas. Three tumors were found to release an homogeneous and acidic (pI = 5.4-4.5) species totally unrelated to the alpha-subunit dissociated from intrapituitary TSH (5 isoforms, pI = 8.8-5.8) while another was more heterogeneous (pI = 8.8, 8.4, 7.6, 6.8, 5.8, 5.4-4.5). Tumoral TSH exhibited at least six detectable isoforms (pI = 8.6, 8.3-8.0, 7.5, 7.0, 6.5, 6.0) very similar to those present in a purified intrapituitary hormone preparation. While intrapituitary TSH was composed of 70% of alkaline (pI = 8.6-7.5), 25% of neutral (pI = 7.0-6.0) and 5% (pI = 5.8-4.5) of acidic forms, these species were found to be more evenly distributed in adenomatous secretion (43%/42%/15%). The TSH-secreting tumors thus appeared to relase preferentially neutral and acidic forms of TSH than alkaline components but for one tumor, this ratio could be modified by chronic incubation with TRH. When assayed for their capacity to stimulate 3H-thymidine incorporation in FRTL-5 cells, neutral TSH appeared definitely less potent than the alkaline and acidic isohormones. Altogether, these data show that pituitary adenomas synthesize normal forms of TSH but release them in variable amount in the medium. When circulating in the blood, the ratio between active and inactive isoforms of TSH may thus be responsible for the variable stimulation of the thyroid gland observed in the patients.

Comparative mapping of human thyrotropin, gonadotropins, and free subunits with antipeptide antibodies.

To compare the structural topology of the human TSH to that of the structurally related gonadotropins, 10 peptides covering the entire primary sequence of the alpha- and beta-subunits of TSH were synthesized and used as antigens for the preparation of polyclonal antibodies. The alpha-subunit was synthesized as 4 nonoverlapping peptides (1-25, 26-51, 49-73, 72-92) while the beta-subunit was segmented in 6 overlapping sequences (2-18, 10-38, 31-51, 53-76, 77-96, 92-112). Most of the peptide sequences were predicted to contain a putative antigenic determinant. All antipeptide antisera were found to bind to the corresponding synthetic sequence in an enzyme-linked immunosorbent assay as well as to denatured TSH subunits after Western blotting. The N-terminal half of the alpha-subunit was found differentially accessible in TSH and gonadotropins compared to the free subunit: antipeptide-alpha 1-25 antibodies exhibited variable affinity for the four glycoprotein hormones whereas anti-alpha 26-51 displayed a remarkable recognition of free alpha-subunit. Four peptides proved to be accessible in the TSH beta-subunit: the N-terminal peptide (beta 2-18) elicited antibodies that bound to free TSH-beta and poorly to the dimer while antibodies against the C-terminal sequence (beta 92-112) recognized equally well free beta-subunit and TSH. Antipeptide-beta 31-51 antibodies proved to be specific for TSH while the beta 53-76 contiguous peptide appeared accessible in both TSH and gonadotropins. The current findings therefore demonstrate that most of the sequences predicted to contain antigenic sites in the alpha- or the beta-subunits are indeed accessible at the surface of these proteins. Additionally, both subunits appear to contain amino acid sequences that are differentially expressed in TSH and gonadotropins as well as in free and combined subunits.

Prolactin isoforms secreted by human prolactinomas.

Prolactin (hPRL) secreted by human prolactinoma cells in culture was purified by gel filtration, lectin affinity chromatography and gel electrophoresis in order to identify the different isoforms of the hormone and to test their respective immunoreactivities and bioactivities. The nonglycosylated hPRL (NG-hPRL), unbound to lectins, was the major form and was a species (NG1-hPRL), of 23,000 (M(r)) apparent molecular weight. The lectin-bound glycosylated hPRL (G-hPRL) consisted of three forms, G1-, G2- and G3-hPRL, of identical molecular weights (25,000 M(r)). Endoglycosidase treatment indicated that these three forms differed by the heterogeneity of their carbohydrate chains. These G-PRLs proved to be 68% less immunoreactive and 50% less bioactive than NG-hPRL. It is concluded from these data that, in prolactinomas, the main variant of the hormone is the nonglycosylated form of PRL.

Evaluation of 29 monoclonal and polyclonal antibodies used in the diagnosis of pituitary adenomas. A collaborative study from pathologists of the Club Français de l'Hypophyse.

Fifteen polyclonal antibodies (pAbs) and 14 monoclonal antibodies (mAbs) directed against hGH, hPRL, beta hFSH, beta hLH, beta hTSH and alpha-subunit were assessed by five different laboratories on normal and adenomatous pituitary tissues. This study aims at providing pathologists with a selected panel of antisera suitable for diagnosis, and appreciating the interest of the recently introduced mAbs. All the anti-hGH Abs proved to be specific (3 pAbs and 4 mAbs); three mAb out of four gave a few false-negative reactions. Three out of six polyclonal anti-hPRL showed cross-reactivity with hGH; anti-hPRL mAbs gave a strong staining with no false-negativity detected so far. MAbs proved to be more efficient for detecting glycoprotein hormones and alpha subunit than pAbs, which, in several cases, gave widespread cross-reactivity. This lack of specificity could explain the noticeable discrepancies reported so far in the appraisal of gonadotropic and somatoprolactinic adenomas.

Immunoreactive and bioactive isoforms of human thyrotropin.

Isoforms of intrapituitary human TSH were separated by gel isoelectrofocusing, and their immunoreactivity analyzed by subsequent immunoblotting using polyclonal and monoclonal antibodies. Under these conditions, TSH polymorphism could be resolved as seven major isoforms (pI 8.6, 8.3, 8.0, 7.5, 7.0, 6.5, and 6.0) by both silver staining of the gels and binding to anti-TSH polyclonal antibodies. The distribution pattern of these forms appeared totally distinct from that of individual TSH alpha (pI 8.8, 8.4, 8.2, 7.6, 7.4, 6.8, 6.6, 5.8, and 5.4) and TSH beta (pI 8.7, 8.1, 7.2, 6.8, 6.2, and 5.8) subunits. While most anti-TSH polyclonal antibodies recognized neutral and alkaline isoforms of TSH (pI 8.6, 8.3, 8.0, 7.5, 7.0, 6.5, and 6.0) through beta determinants, they displayed a variable potency to bind acidic forms of the hormone (pI 5.8, 5.5, 4.8, and 4.5), in contrast to anti-TSH alpha antisera, which enlighted the broadest spectrum of isoforms. Monoclonal antibodies of various specificities largely reproduced this distribution, indicating that at least five distinct epitopes are coexpressed in the neutral and alkaline forms of TSH, but only two are expressed in the acidic ones. All of the forms were found to induce cAMP production and stimulate growth of FRTL-5 rat thyroid cells, although neutral forms proved to be definitely less potent than the others. We therefore, conclude that TSH isoforms differ in the expression of both their immunoreactive and bioactive domains and that the bioactive/immunoreactive ratio is not an accurate index for the biopotency of the hormone.

Differential effect of glycosylation on the expression of antigenic and bioactive domains in human thyrotropin.

Enzymatic deglycosylation of human thyroid-stimulating hormone (hTSH) was shown to result in a mixture of partially and fully deglycosylated forms of the hormone by gel electrophoresis, silver staining and immunoblotting. Radioiodination of the enzymatic digest, followed by gel filtration and concanavalin A-Sepharose chromatography allowed to separate two different forms of partially deglycosylated [125I]hTSH and a fully deglycosylated hormone. The final recovery was of approx. 60% for [125I]hTSH deglycosylated in its beta-subunit, of 30% for [125I]hTSH missing the oligosaccharide in beta and one in alpha but only of 10% for [125I]hTSH deglycosylated in both the alpha- and beta-subunits. Gel electrophoresis under non-denaturing conditions showed that each form migrated distinctly from free subunits and reverse-phase high performance liquid chromatography after reduction and carboxymethylation identified the presence of the two subunits. Mapping of [125I]hTSH derivatives with polyclonal, monoclonal and anti-peptide antibodies allowed to identify two novel glycosylation-independent epitopes preserved in deglycosylated hTSH while the main immunogenic determinant was lost. When assayed in a bioassay with FRTL-5 cells, the hormone deprived of its beta-linked carbohydrate chain was found to be as effective as the native hormone on cAMP production and cell growth. In contrast, the fully deglycosylated derivative proved to stimulate cAMP release but appeared to be definitely less potent on thyroid cell growth. Our findings thus demonstrate that glycosylation of the alpha-subunit but not that of the beta-subunit is essential to express the domains involved in hTSH immunoreactivity as well as those controlling the post-receptor biological activity of the hormone.