Uterine length and fertility outcomes: a cohort study in the IVF population.

STUDY QUESTION: What is the relationship between pre-cycle uterine length and IVF outcome (chemical pregnancy, clinical pregnancy, spontaneous abortion and live birth)? SUMMARY ANSWER: Women at extremes of uterine length (<7.0 or >9.0 cm) were less likely to achieve live birth and women with uterine lengths <6.0 cm were also more likely to experience spontaneous abortion. WHAT IS KNOWN ALREADY: A prospective study of 807 women published in 2000 found that implantation and clinical pregnancy rates were highest in women with uterine lengths between 7.0 and 9.0 cm, though the difference was not significant. The relationship between pre-cycle uterine length and live birth has not been evaluated. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study of all cycles performed after uterine length measurement at an academic hospital IVF clinic from 2001 to 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 8981 fresh cycles were performed in 5120 adult women with normal uterine anatomy. Women with uterine anomalies (unicornuate, bicornuate, septate or uterus exposed to diethylstilbestrol) were excluded and women with fibroids were identified for subanalysis. Uterine length was measured by uterine sounding. Cycles were divided by uterine length into groups: <6.0 cm (very short, n = 76), 6.0-6.9 cm (short, n = 2014), 7.0-7.9 cm (referent, n = 4984), 8.0-8.9 cm (long, n = 1664) and ≥9 cm (very long, n = 243). Multivariate logistic regression (first-cycle analyses) and generalized estimating equations (all-cycle analyses) were adjusted for age, fibroids and ART treatment (assisted hatching, intracytoplasmic sperm injection) to generate relative risk (RR) of cycle outcomes by uterine length. MAIN RESULTS AND THE ROLE OF CHANCE: Median uterine length in the IVF population was 7.0 cm (interquartile range 7.0-7.8) and was positively associated with BMI (P < 0.001) and fibroids (P = 0.02). Compared with the referent group, women with uterine lengths <6.0 cm were half as likely to achieve live birth (RR: 0.53; 95% confidence interval (CI): 0.35-0.81) and women with lengths of 6.0-6.9 cm were also less likely (RR: 0.91; CI: 0.85-0.98). Cubic regression spline identified a significant inverse U-shaped association whereby women with uterine lengths <7.0 or >9.0 cm were less likely to achieve live birth. Women with lengths <6.0 cm were also more likely to experience spontaneous abortion (RR: 2.16; CI: 1.23-3.78). Results remained consistent when excluding women with a uterine factor diagnosis (n = 8823), when limiting to the first cycle at our institution (n = 5120) and when further restricting to first-ever cycles (n = 3941). LIMITATIONS, REASONS FOR CAUTION: Optimal assessment of uterine length by ultrasound was not feasible due to time and cost limitations, though uterine sounding is a clinically relevant measurement allowing for results with practical implications. Findings from our predominantly Caucasian clinic population may not be generalizable to infertile populations with different ethnic compositions. WIDER IMPLICATIONS OF THE FINDINGS: Reproducibility of results would solidify findings and inform patient counseling in women undergoing IVF. STUDY FUNDING/COMPETING INTEREST(S): No funding was sought for this investigation. MD declares relationships with UpToDate (royalties) and WINFertlity (consultant).

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the 6.7 K/gp19 K region.

The use of genetically engineered, replication-selective viruses to treat cancer is being realized with viruses such as ONYX-015, a human adenovirus that selectively destroys p53 mutant cancer cells. To enhance further the clinical efficacy of ONYX-015 and viruses like it, we have developed a novel gene delivery system for replicating adenoviruses. This system has two unique features. First, it uses the endogenous adenoviral gene expression machinery (promoter, splicing, polyadenylation) to drive transgene expression. Second, a single region or gene in the multi-gene E3 transcription unit is selectively substituted for by the therapeutic transgene(s). Analyzing various transgene substitutions for the 6.7 K/gp19 K region of E3, we demonstrate the following: (1) transgene expression in this system is predictable and mimics the substituted endogenous gene expression pattern, (2) expression of surrounding E3 genes can be retained, (3) the insertion site choice can effect both the transgene expression level and the viral life cycle, and, (4) expression levels from this system are superior to those generated from a replication-defective virus using the HCMV enhancer-promoter and this is dependent on viral DNA replication. This unique methodology has broad application to the rapidly evolving field of replicating virus-based therapies.

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the ADP region.

Genetically modified replication-selective human adenoviruses are currently undergoing testing in the clinical setting as anticancer agents. Coupling the lytic function of these viruses with virus-mediated transgene delivery represents a powerful extension of this treatment. We have designed a unique system for gene delivery from the replicating virus. It takes advantage of the endogenous gene expression control sequences (promoter, splicing, polyadenylation signals) to efficiently and predictably deliver transgenes from the non-essential E3 transcription unit while still maintaining the expression of the remaining E3 genes in the multi-gene transcription unit. In this article, we engineered restriction enzyme sites into the virus genome selectively to delete the ADP gene and replace it with the therapeutic transgenes CD and TNFalpha. We demonstrate that: (1) transgene expression from this region mirrors the substituted ADP gene; (2) the loss of ADP in these viruses results in infected cells with extended viability and protein synthesis when compared with a wild-type Ad5 infected cell; and (3) expression of surrounding E3 genes can be maintained in such a system. The potential advantages of delivering transgenes from the ADP region of the replicating adenovirus are discussed.

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the E3B region.

Successful therapies for cancer need to deal with the complexity associated with the human tumor. Studies of tumor and viral biology have progressed to a point where replicating viruses are now being engineered as potential treatments for human cancers. The complex nature of human cancers dictates that successful treatments will require combination therapies. To this end, we have focused on developing the gene delivery capacity of the replicating adenovirus, using the non-essential E3 region transcription unit as a target site for therapeutic transgene insertions. Utilizing the endogenous expression machinery of the E3 region (promoter, splicing, polyA) we show that a therapeutic transgene, TNF, is efficiently expressed from the E3B region and with exclusive late gene expression kinetics. Potential clinical applications are discussed.

Region E3 of subgroup B human adenoviruses encodes a 16-kilodalton membrane protein that may be a distant analog of the E3-6.7K protein of subgroup C adenoviruses.

There is an open reading frame in the E3 transcription unit of adenovirus type 3 (Ad3) and Ad7 that could encode a protein of 16 kDa (16K protein). Ad3 and Ad7 are members of subgroup B of human adenoviruses. Using a rabbit antipeptide antiserum, we show that the 16K protein is expressed in Ad3- and Ad7-infected cells at early and late stages of infection; it is not expressed in cells infected with an Ad7 mutant that deletes the 16K gene. The 16K protein was also transcribed and translated in vitro from DNA containing the open reading frame for the 16K protein. The 16K protein has two hydrophobic domains typical of integral membrane proteins; consistent with this, we detected 16K in the crude membrane but not the cytosol cellular fractions. Although 16K has two potential sites for Asn-linked glycosylation, the protein is not glycosylated. The 16K gene is located in the same position in region E3 as the gene for the 6.7K protein of subgroup C adenoviruses (Ad2 and Ad5). E3-6.7K is an Asn-linked integral membrane glycoprotein, localized in the endoplasmic reticulum, whose function is unknown. The 16K protein has a putative transmembrane domain located in the same place in 16K as is the transmembrane domain in 6.7K, and the C-terminal portion of 16K is partially homologous to the C-terminal cytoplasmic domain of 6.7K; we suggest that these domains in 16K and 6.7K may have a similar function. The N-terminal 102 residues in 16K are not found in 6.7K; these residues may have a function that is unique to the 16K protein. In common with all known E3 proteins, the 16K protein is dispensable for virus replication in cultured cells; this suggests that the 16K protein may function in virus-host interactions.

The E3-20.5K membrane protein of subgroup B human adenoviruses contains O-linked and complex N-linked oligosaccharides.

Subgroup B adenoviruses (Ad3, -7, -11, -35) contain two open reading frames (ORFs) in the early E3 transcription unit that are not present in subgroup C adenoviruses (Ad2, Ad5). The product of one of these ORFs, a 20,500-kDa (20.5K) protein, was shown previously to be expressed as two diffuse 22K and 36K bands on SDS-PAGE; the 22K appeared to be the precursor to the 36K species. As judged by its predicted sequence, 20.5K is a type I membrane glycoprotein with two potential sites for N-glycosylation and a transmembrane domain near its COOH-terminus. Here we show that when Ad3- or Ad7-infected cells were radiolabeled in the presence of tunicamycin, which prevents the addition of N-linked oligosaccharides, both the 22K and the 36K forms of 20.5K showed increased mobility in SDS-PAGE, indicating that both forms contain N-linked sugars. Both the 22K and the 36K forms were sensitive to digestion by endoglycosidase F and N-glycanase, again indicating that they both contain N-linked sugars. Only the 22K species was sensitive to endoglycosidase H, indicating that it contains high-mannose-type oligosaccharides and that the 36K species contains complex-type carbohydrates. The 36K form was sensitive to neuraminidase, indicating that its sugars contain terminal sialic acid. When digested with N-glycanase and neuraminidase, the 36K form was sensitive to O-glycanase, indicating that the 36K form has O-linked oligosaccharides. The 22K form was labeled with [3H]mannose and the 36K form was labeled with [3H]glucosamine and to a much lesser extent by [3H]mannose. Altogether these results indicate that the 20.5K protein is cotranslationally modified with N-linked high-mannose oligosaccharides, then the protein moves into the Golgi and trans-Golgi network where it acquires O-linked and complex N-linked oligosaccharides.

A 20,500-Dalton protein is coded by region E3 of subgroup B but not subgroup C human adenoviruses.

There is an open reading frame (ORF) between ATG2089 and TGA2656 in the early E3 transcription unit of subgroup B adenovirus 3 (Ad3) that could encode a protein of 20,500 MW (20.5K). This ORF also exists in Ad7, Ad11, and Ad35 (subgroup B). An antipeptide antiserum made in rabbits against the predicted Ad3/7 20.5K protein immunoprecipitated two diffuse bands with apparent molecular weights of about 22K and 36K from Ad3- or Ad7-infected cells. These bands were also detected in immunoblots. These bands were not seen in mock-infected cells or cells infected with Ad3 or Ad7 mutants that delete the gene for 20.5K. In vitro transcription and translation of the 20.5K gene yielded a protein of about 18K, suggesting that this may be the primary translation product and that the 22K and 36K forms of 20.5K arise by post-translation processing. Pulse/chase experiments suggest that the half-life of the 22K form is short, and that this form is further modified to the 36K species. In accord with these results and as judged by its predicted sequence, 20.5K appears to be a membrane glycoprotein with a potential N-terminal signal sequence, a second hydrophobic putative transmembrane domain, and two potential Asn-linked glycosylation sites. The 20.5K protein was synthesized throughout the course of the infection. Ad3 and Ad7 mutants lacking 20.5K grew as well as wild-type Ad3 and Ad7, indicating that, in common with subgroup C E3 proteins, the 20.5K protein is dispensable for virus replication in cultured cells. The 20.5K gene is totally absent from the E3 region of Ad2 and Ad5 (subgroup C). The gene is also absent or highly diverged in the E3 region of Ad12 (subgroup A) and Ad40 (subgroup F). Given that E3 genes may counteract host defenses, the 20.5K protein may contribute to the unique pathogenic properties of subgroup B human adenoviruses.

A 12,500 MW protein is coded by region E3 of adenovirus.

There is an open reading frame between ATG291 and TGA612 in the early region E3 transcription unit of adenovirus 2 (Ad2) that could encode a protein of 12,500 MW (12.5K). To address whether this protein is synthesized, we generated an antiserum against a TrpE-12.5K fusion protein which was expressed in Escherichia coli. This antiserum immunoprecipitated a doublet of about 12.5K apparent MW from [35S]Cys-labeled cells infected with Ad2, Ad5, and various mutants in other E3 genes. Mutants in the 12.5K gene did not produce this protein, and an in-frame deletion mutant showed a protein with a corresponding decrease in size. Cell-free translation of hybridization-purified RNA indicated that 12.5K is coded by E3 mRNA i. mRNA i is relatively scarce, and 12.5K is synthesized in correspondingly small amounts. The 12.5K protein was synthesized at early and late stages of infection in comparable amounts. Pulse-chase experiments indicated that 12.5K has a half-life of about 10 hr. The function of 12.5K is unknown, and the 12.5K gene can be deleted without affecting virus growth in cell culture. However, 12.5K is likely to be important in vivo because the gene is highly conserved in both Ad2 and Ad5 (group C adenoviruses), and also in Ad3 (group B).

Presence of an intron in elongator methionine-tRNA of Halobacterium volcanii.

The gene for the elongator (internal) methionine transfer RNA (tRNA(mMet)) in the archaebacterium Halobacterium volcanii contains a 75 base pair intron (intervening sequence) and lacks the 3'-terminal CCA sequence of the mature tRNA. There is a single copy of this gene in the genome and it is expressed. Northern hybridization experiments indicate that the precursor is processed to produce mature tRNA and a free intron species. A secondary structure of the transcript can be formed in which the anticodon stem of the tRNA is extended. Two symmetrically placed three-base bulge loops separated by a 4 base pair stem are present in this extension. The cleavage sites for the removal of the intron are placed between the middle and 3' residues of these loops.