A randomized controlled trial of AMH-based individualized FSH dosing in a GnRH antagonist protocol for IVF.

STUDY QUESTION: Does an individualized serum anti-Müllerian hormone (AMH) based FSH dosing algorithm used in a GnRH antagonist protocol increase the proportion of patients with an intended number of oocytes (5-14) retrieved compared with a standard regimen? SUMMARY ANSWER: The AMH-based individualized algorithm did not increase the proportion of patients with an intended oocyte retrieval. WHAT IS KNOWN ALREADY: Individualizing treatment for ovarian stimulation by serum AMH or antral follicle count can theoretically improve the ratio between benefits and risks. Current data suggest that there may be a reduced risk of ovarian hyperstimulation syndrome (OHSS), but without improved pregnancy or live birth rates. Only two randomized controlled trials (RCTs) have examined the potential of AMH-based algorithms to optimize the FSH dosing in ovarian stimulation. STUDY DESIGN SIZE DURATION: A dual-center open-label investigator-driven RCT was conducted between January 2013 and November 2016. Eligibility was assessed in 269 women and 221 were randomized 2:1 between individualized and standard dosing groups. Women with pretreatment serum AMH > 24 pmol/L had 100 IU/day of recombinant FSH (rFSH); AMH 12-24 pmol/L had 150 IU/day of rFSH, and AMH < 12 pmol/L had maximal stimulation with corifollitropin 100 or 150 mg depending on bodyweight ±60 kg. The standard group had 150 IU/day of rFSH irrespective of pretreatment AMH. All patients followed the GnRH-antagonist protocol.The sample size calculation assumed that individualized dosing by AMH would reduce the proportion of unintended oocyte yield (outside the 5-14 range) by 50%, from 35 to 17.5%. In a 2:1 randomization this required 216 patients: 144 in the individualized and 72 patients in the standard group (80% power, 5% significance). PARTICIPANTS/MATERIALS SETTING METHODS: All women had a presumed ovulatory normal menstrual cycle, were aged 25-38 years, weighed < 75 kg, had pretreatment AMH 4-40 pmol/L, did their first IVF or ICSI cycle and had two ovaries accessible to oocyte retrieval. Recruitment was conducted from both participating sites. Women were excluded if diagnosed with anovulatory polycystic ovary syndrome, endometriosis grade III/IV, hydrosalpings on ultrasound, recurrent miscarriages (≥3), FSH > 12 IU/L or major medical disorders. MAIN RESULTS AND THE ROLE OF CHANCE: After randomization 149 women were allocated to the individualized group and 72 to the standard group. The primary outcome of women with an intended (5-14) number of oocytes retrieved was similar in the individualized (n = 105) versus the standard (n = 55) rFSH treatment group (72% [95% CI 64-79%] versus 78% [95% CI 67-86%], respectively, P = 0.68, between group standardized mean difference (SMD) -6%, 95% CI: -19-8%). In the high AMH stratum of the individualized group, significantly more women (n = 13) had an unintended low number of oocytes (<5) retrieved (38% [95% CI: 23-55%]) compared with the standard group (6% [95% CI 0.3-24%], P = 0.029, between group SMD 32%, 95% CI: 9-56%). Conversely, in the low pretreatment AMH stratum, individualized dosing using corifollitropin reduced the proportion of unintended low responders to 24% (95% CI: 12-40%) compared with 47% (95% CI: 26-69%) in the standard group, P = 0.10, between group SMD -23% (95% CI: -54-8%). OHSS was diagnosed in four women (two in each study arm), and all cases were mild. Daily luteal phase questionnaire reporting showed similar wellbeing in terms of abdominal distention, abdominal pain, dyspnea and occurrence of bleeding between groups. The cumulative live birth rate per started cycle was similar (32 and 35%) comparing the individualized with the standard group. LIMITATIONS REASONS FOR CAUTION: This study was powered for showing differences only in the distribution of oocyte retrieval when comparing individualized and standard groups, therefore additional results should be viewed with caution. In addition, there was a change of AMH assay halfway through the study period and the possibility that corifollitropin being introduced to a subgroup of the intervention has introduced confounding cannot be ruled out. WIDER IMPLICATIONS OF FINDINGS: In the expected high responder AMH stratum, 100 IU/day is an insufficient rFSH dose in a high proportion of patients. Further research might explore the 125 IU/day dose for the high AMH segment. STUDY FUNDING/COMPETING INTERESTS: None for the submitted work. ICMJE declared personal interests for two of the authors. TRIAL REGISTRATION NUMBER: EUDRACT registration number: 2012-004969-40. TRIAL REGISTRATION DATE: 27 November 2012. DATE OF FIRST PATIENT'S ENROLLMENT: 10 January 2013.

Association of Streptococcus pluranimalium with valvular endocarditis and septicaemia in adult broiler parents.

The genus Streptococcus consists of more than 60 species, but only Streptococcus equi subspecies zooepidemicus, Streptococcus gallolyticus ssp. gallolyticus, Streptococcus gallinaceus, Streptococcus dysgalactiae, Streptococcus mutans and Streptococcus suis have been isolated from poultry. During investigations of the aetiology of increased mortality in broiler parent stock at the end of production, pure cultures of streptococcal-like organisms that could not be classified among these six species were obtained from 24 cases of septicaemia or valvular endocarditis and septicaemia. Phenotypic characterization using the API20 STREP kit identified the isolates as Aerococcus viridans (10), Aerococcus urinae (2), Leuconostoc species (4), Streptococcus salivarius (2), Streptococcus bovis II 3 (1), Enterococcus avium (3), Enterococcus faecium (1) or Gemella morbillorum (1). However, this identification was misleading as subsequent genetic investigations using pulse field gel electrophoresis and sequencing of 16S rRNA genes showed that 19 isolates were classified as Streptococcus pluranimalium, while the remaining isolates were E. avium (3), E. faecium (1) or Lactobacillus species (1). Misidentification by API20 STREP was related to the database provided by the manufacturer, as the phenotypic characteristics could identify these organisms as S. pluranimalium. The isolates of S. pluranimalium belonged to at least three different clones as determined by pulsed field gel electrophoresis of SmaI-digested genomic DNA. The capacity that these isolates had to colonize the valvular endothelium was suggested by the occurrence of valvular endocarditis in 12 of 19 cases. Demonstration of the same clone in all four houses on a farm suggested the pathogenic potential of this organism.

Nucleotide sequence of a beta-1,3-glucanase isoenzyme IIA gene of Oerskovia xanthineolytica LL G109 (Cellulomonas cellulans) and initial characterization of the recombinant enzyme expressed in Bacillus subtilis.

The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).

Molecular cloning of a lytic beta-1,3-glucanase gene from Oerskovia xanthineolytica LLG109. A beta-1,3-glucanase able to selectively permeabilize the yeast cell wall.

Molecular cloning of the beta gIII gene encoding for an endo-beta-1,3-glucanase (beta gl II) from Oerskovia xanthineolytica LLG109, a yeast-lytic gram-positive bacterium, has been conducted in order to elucidate its primary sequence and subsequently express it into B. subtilis. This endo-beta-1,3-glucanase exhibits low yeast-lytic activity toward viable S. cerevisiae cells, and it has shown ability to selectively permeabilize the yeast cell wall and release intracellular proteins produced by yeast. Highly degenerate oligonucleotides have been used to PCR-amplify a region of the beta-1,3-glucanase II encoding gene from O. xanthineolytica LLG109. The amplified fragment has been cloned and sequenced. The deduced amino acid sequence contains regions identical to the amino acid sequences previously determined by direct sequencing of the purified enzyme from O. xanthineolytica LLG109. By using the 180-bp PCR product as a homologous probe, we have been able to isolate four positive clones harboring plasmids pPF1A, pPF1B, pPF8A, and pPF9A, respectively, from a partial genomic library from O. xanthineolytica LLG109. All four plasmids contained a 2.7-kb BamHI insert that hybridized to the PCR probe under high stringency conditions. The 2.7-kb fragment seemed to be identical in all four cases regarding preliminary partial restriction mapping analysis done on the four plasmids. The 1.5-kb BamHI/KpnI restriction fragment from pPF8A and pPF9A hybridizing with the 180-bp PCR probe is presently being sequenced. The cloning of the lytic beta-1,3-glucanase from O. xanthineolytica LLG109 expands the number of yeast lytic beta-glucanases so far cloned. The availability of the nucleotide sequences of such a family of genes will allow further understanding of the role and mode of action of these enzymes in yeast cell wall degradation. In addition, a more extensive study on the structure and functional relationships of these enzymes will allow us to engineer "tailor-made" lytic beta-1,3-glucanases for use in new and improved large-scale selective cell permeabilization (SCP) and selective protein recovery (SPR) from yeast cells, not only from S. cerevisiae but also from alternative yeast expression systems such as Hansenula polymorpha, Pichia pastoris, and others, which are becoming of increasing importance in biotechnology.

Protein engineering of subtilisins to improve stability in detergent formulations.

Microbial proteases are used extensively in a large number of industrial processes and most importantly in detergent formulations facilitating the removal of proteinaceous stains. Site-directed mutagenesis has been employed in the construction of subtilisin variants with improved storage and oxidation stabilities. It is shown that in spite of significant structural homology between subtilisins subjected to protein engineering the effects of specific mutations can be quite different. Mutations that stabilize one subtilisin may destabilize another.

Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis.

A gene for alpha-acetolactate decarboxylase (ALDC) was cloned from Bacillus brevis in Escherichia coli and in Bacillus subtilis. The 1.3-kilobase-pair nucleotide sequence of the gene, aldB, encoding ALDC and its flanking regions was determined. An open reading frame of 285 amino acids included a typical N-terminal signal peptide of 24 or 27 amino acids. A B. subtilis strain harboring the aldB gene on a recombinant plasmid processed and secreted ALDC. In contrast, a similar enzyme from Enterobacter aerogenes is intracellular.

The major subunit of Escherichia coli type 1 fimbriae is not required for D-mannose-specific adhesion.

Type 1 fimbriae are surface organelles on Escherichia coli, which mediate specific binding to D-mannose-containing structures. These fimbriae are heteropolymers composed of a major building element, the FimA protein, and small amounts of the FimF, FimG and FimH proteins. The FimH protein is uniquely responsible for the D-mannose receptor binding. In this work data are presented which indicate that the major subunit of type 1 fimbriae is dispensable for D-mannose-specific binding. A recombinant strain was studied which harboured an insertional deletion in the fimA gene, and was thereby unable to produce type 1 fimbriae; however, it was still able to express a D-mannose-binding phenotype. However, the deletion resulted in a 25-fold reduction of the adhesive potential, as measured by binding to D-mannose-coated Sepharose beads. Serological and specific receptor binding evidence is presented that suggests that the FimH adhesion is capable of being exposed on the bacterial surface without being an integral part of the fimbriae.

Type 1 fimbriae of Escherichia coli as carriers of heterologous antigenic sequences.

A strategy has been designed for the construction of recombinant bacterial strains which eventually may become useful as live vaccines and which may also be relevant for the preparation of conventional vaccines. The approach used is the fusion of small antigenic peptide sequences into specific segments of a protein whose location on the bacterial surface ensures that the recombinant organism is able to present the inserted antigen to the host (animal or human) infected by the bacterium. The chosen surface protein is a naturally occurring polymer of Escherichia coli, viz., type 1 fimbriae. The results obtained show that fusion of such foreign sequences into selected points of the structural protein of the fimbriae results in the production of functionally normal type 1 fimbriae. Furthermore, hybrid fimbriae carrying such small epitope sequences can be recognized by antibodies directed against the foreign parent protein. This observation is an important prerequisite for the eventual design of useful vaccines. The analysis of the fimbrial protein and its potential as a carrier of foreign peptides from hepatitis B surface antigen, foot-and-mouth disease virus and poliovirus indicated that there may be several positions in the protein which may turn out to be relevant for this purpose and be important fusion sites.

Blood lead-blood pressure relations: alcohol intake and hemoglobin as confounders.

A cohort of 1,052 persons (504 men and 548 women) born in 1936 and residing in the Glostrup area, Denmark, underwent a comprehensive physical examination in 1976 at age 40 years, and 966 underwent a complete reexamination five years later. The examinations included blood lead concentration and blood pressure assessment under careful quality control. Complete blood lead and blood pressure data were available for 861 of these subjects (451 men and 410 women). The median blood lead levels were 13 and 9 micrograms/100 ml at age 40 years and 9 and 6 micrograms/100 ml at age 45 years in men and women, respectively. A slightly increased blood lead concentration was seen at age 40 years in women with a systolic blood pressure above 140 mmHg and/or a diastolic blood pressure above 90 mmHg. Systolic blood pressure in men and women and diastolic blood pressure in women correlated significantly with log blood lead at age 40 years but not at age 45 years, a doubling in blood lead being associated with an increase in blood pressure of 3 mmHg or less. Of nine potential confounders assessed, only blood hemoglobin and alleged alcohol intake were significantly associated with both blood lead and blood pressure. If one or both confounders were entered into a multiple regression analysis, all associations between blood lead and blood pressure became nonsignificant, in some cases with a negative regression coefficient. In addition, the blood lead:hemoglobin ratio was poorly associated with blood pressure, particularly in individuals with a low alcohol intake. Because both hemoglobin level and alcohol intake appear to be biologically plausible confounders, any independent effect of low-level lead exposure on blood pressure could not be determined.

Symptoms and signs of polyneuropathy and their relation to alcohol intake in a normal male population.

In a normal population of 468 45-year-old males symptoms and signs of polyneuropathy were investigated and related to self-reported alcohol consumption and associated problems. Sensory disturbances in the feet were claimed by 3.4%, 5.8% had abolished Achilles tendon reflexes, and 7.7% increased vibratory perception thresholds. Significant correlations were found between persons with signs of neuropathy and persons with physical or social consequences of alcohol abuse.

Urinary incontinence in 45-year-old women. An epidemiological survey.

In an epidemiological health survey, 515 45-year-old women were interviewed about urological problems, particularly incontinence. A pelvic examination was also conducted on 509 of the women. Twenty-two per cent or 114 women stated that they experienced incontinence, which took the form of stress incontinence in 75%, urge incontinence in 11% and a mixture of the two in 14%. Only 14 women, 3% of all the women interviewed, desired medical treatment for incontinence. In the incontinent women, the pelvic examination significantly more often revealed a cystocele, uterine prolapse or impaired function of the levator muscles. No correlation was found between an enlarged uterus and incontinence. In 211 women with one or more of these findings at the gynaecological examination, the frequency of incontinence was 35%; in 298 women with no pathological findings, the frequency was 15%. The frequency of urinary incontinence was not increased in women with higher parity or in postmenopausal women.

Analysis of the mycoplasma genome by recombinant DNA technology.

A library of DNA fragments from Mycoplasma sp. strain PG50 has been made in the vector pBR325. Analysis in Escherichia coli minicells of randomly picked clones from this library demonstrated that many plasmids can promote synthesis of mycoplasma protein in the E. coli genetic background. Screening with labeled rRNA identified plasmids with sequences from the beginning of the rRNA cistron to about two-thirds within the gene for 23S rRNA. Use of these plasmids as probes allowed an accurate determination of the number of RNA cistrons as two, and demonstrated that the cistrons are more than 20 kbp apart. The DNA sequence of 16S rRNA and the surrounding control regions has been determined.

Sex-dependent relation between ionized calcium in serum and blood pressure.

In an epidemiological study of 977 45-year-olds, both the mean concentration of ionized calcium in serum, [Ca2+]7.4, and arterial blood pressure were higher in men than in women. Among the men, there was a weak positive correlation between [Ca2+]7.4 and the systolic blood pressure, but no other significant correlation was found within each separate sex. Postmenopausal women had slightly increased [Ca2+]7.4 but normal blood pressure. Patients taking thiazide diuretics or beta-adrenergic blocking agents had normal [Ca2+]7.4.