The impact of luteal serum progesterone levels on live birth rates-a prospective study of 602 IVF/ICSI cycles.

STUDY QUESTION: Is the chance of a live birth following IVF treatment and fresh embryo transfer affected by early and mid-luteal serum progesterone (P4) levels? SUMMARY ANSWER: Low as well as high serum P4 levels in the early and mid-luteal phase reduce the chance of a live birth following IVF treatment with fresh embryo transfer. WHAT IS KNOWN ALREADY: Data from non-human studies and studies of frozen-thawed embryo transfer cycles indicate that low as well as high P4 levels during the mid-luteal phase decrease the chance of pregnancy. The altered P4 pattern may disrupt the endometrial maturation leading to asynchrony between embryonic development and endometrial receptivity, thereby, compromising implantation and early development of pregnancy. STUDY DESIGN, SIZE, DURATION: Prospective multicenter cohort study of 602 women undergoing IVF treatment. Patients were recruited from four Danish public Fertility Centers from May 2014 to June 2017. The study population was unselected, thus, representing a normal everyday patient cohort. Patients were treated in a long GnRH-agonist protocol or a GnRH-antagonist protocol and triggered for final oocyte maturation with either hCG or a GnRH-agonist. The same vaginal luteal support regimen was applied in all patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Serum P4 levels from the early or mid-luteal phase were correlated to positive hCG and live birth rates (delivery > gestational week 20). Patients were divided into four P4 groups based on raw data of P4 serum levels and reproductive outcomes during early luteal phase (P4<60 nmol/l, P4 60-100 nmol/l, P4 101-400 nmol/l and P4>400 nmol/l) and during mid-luteal phase (P4<150 nmol/l, P4 150-250 nmol/l, P4 251-400 nmol/l and P4>400 nmol/l). MAIN RESULTS AND THE ROLE OF CHANCE: The optimal chance of pregnancy was achieved with serum P4 levels of 60-100 nmol/l in the early luteal phase whereas the optimal P4 level during the mid-luteal phase was 150-250 nmol/l. Below, but most distinctly above these levels, the chance of pregnancy was consistently reduced. With an early luteal P4 level of 60-100 nmol/l, the chance of a positive hCG-test was 73%, 95% CI: [59, 84] following cleavage stage embryo transfer. In contrast, with P4 levels >400 nmol/l, the chance of a positive hCG-test was significantly reduced to 35%, 95% CI: [17, 57], thus, an absolute risk difference of -38%, P = 0.01. A similar negative association between early luteal P4 and live birth rate was found, although it did not reach statistical significance. During the mid-luteal phase, a P4 level of 150-250 nmol/l resulted in an optimal chance of live birth: 54%, 95% CI: [37, 70] compared to 38%, 95% CI: [20, 60] with a P4 level >400 nmol/l, thus, an absolute risk difference of -16%, P = 0.14. All estimates were adjusted for maternal age, maternal BMI, study site, final follicle count and late follicular P4 levels. LIMITATIONS, REASONS FOR CAUTION: This study is the first to explore the possible upper and lower thresholds for luteal P4 following IVF treatment and fresh embryo transfer, and the optimal P4 ranges found in this study should be corroborated in future clinical trials. Furthermore, the P4 thresholds in this study only apply to fresh IVF cycles, using vaginal luteal phase support, as the optimal P4 level in cycles using intramuscular P4 may be different. WIDER IMPLICATIONS OF THE FINDINGS: Future studies are necessary to explore whether additional exogenous luteal P4 supplementation in the low P4 group could increase the chance of a live birth following fresh embryo transfer, and whether patients with luteal P4 levels >400 nmol/l would benefit from segmentation followed by subsequent transfer in frozen/thawed cycles. TRIAL REGISTRATION NUMBER: NCT02129998 (Clinicaltrials.gov). STUDY FUNDING/COMPETING INTEREST(S): L.H.T. received an unrestricted grant from Ferring Pharmaceuticals, Denmark, to support this study. P.H. received unrestricted research grants from MSD, Merck, Gedeon Richter and Ferring Pharmaceuticals outside of this work as well as honoraria for lectures from MSD, Merck and Gedeon Richter outside of this work. U.K. received honoraria for lectures from MSD and Ferring Pharmaceuticals outside of this work. C.A. received unrestricted research grants from MSD, IBSA, and Ferring Pharmaceuticals outside of this work as well as honoraria for lectures from MSD and IBSA. H.O.E. and B.B.P. received an unrestricted research grant from Gedeon Richter outside of this work. K.E., L.B., D.P. and B.H. have no conflict of interest. Furthermore, grants from 'The Health Research Fund of Central Denmark Region', 'The Research Foundation of the Hospital of Central Jutland', 'The Research Foundation of A.P. Møller', 'The Research Foundation of Aase & Ejnar Danielsen', 'The Research Foundation of Dagmar Marshall', 'The Research Foundation of Dir. Jacob Madsen & Hustru Olga Madsen', 'The Research Foundation of Fam. Hede Nielsen' and 'The Danish Medical Research Grant' supported conducting this study. The providers of funding were neither involved in the conduction of the study nor in the writing of the scientific report.

Progesterone supplementation during early gestations after IVF or ICSI has no effect on the delivery rates: a randomized controlled trial.

BACKGROUND: The aim was to study whether prolongation of luteal support during early pregnancy influences the delivery rate after IVF. METHODS: Dual centre study including 303 women who achieved pregnancy after IVF or ICSI was used. All were treated with the long protocol using GnRH agonists and given luteal support with 200 mg vaginal progesterone three times daily during 14 days from the day of transfer until the day of a positive HCG test. The study group (n = 150) withdrew vaginal progesterone from the day of positive HCG. The control group (n = 153) continued administration of vaginal progesterone during the next 3 weeks of pregnancy. RESULTS: The number of miscarriages prior to and after week 7 of gestation was seven (4.6%) and 15 (10.0%) in the study group and five (3.3%) and 13 (8.5%) in the control group respectively. The number of deliveries was 118 (78.7 %) in the study group and 126 (82.4 %) in the control group. The differences were not significant. CONCLUSIONS: Prolongation of progesterone supplementation in early pregnancy has no influence on the miscarriage rate, and thus no effect on the delivery rate. Progesterone supplementation can safely be withdrawn at the time of a positive HCG test.

Zinc supplementation during pregnancy: a double blind randomised controlled trial.

OBJECTIVE: To investigate whether zinc supplementation during pregnancy improves maternal and fetal outcome. DESIGN: Controlled clinical trial started at registration until discharge of mother and child from hospital. Two thousand volunteer mothers were randomly assigned to receive zinc supplementation or placebo in a double blind trial. PATIENTS: Women less than 20 weeks pregnant at the first visit who agreed to take part in the study. One thousand two hundred and six mothers were available for study. INTERVENTIONS: Volunteers were randomly selected to receive two tablets of zinc (44 mg zinc in total) or two placebo tablets containing inert substances, indistinguishable in appearance and taste from the zinc tablets. OUTCOME MEASURE: Large for gestational age (LGA), small for gestational age (SGA), premature rupture of the membrane (PROM), preterm labor (PL), preeclampsia and bleeding in the second or third trimester. RESULTS: There were no differences between mothers given zinc supplementation concerning the outcome parameters and those given placebo. CONCLUSION: Zinc supplementation in pregnancy in a normal healthy middle class population in Denmark does not seem to offer any benefits to the mother or her fetus.

Multiple non-LTR retrotransposons in the genome of Arabidopsis thaliana.

DNA sequence analysis near the Arabidopsis thaliana ABI3 gene revealed the presence of a non-LTR retrotransposon insertion that we have designated Ta11-1. This insertion is 6.2 kb in length and encodes two overlapping reading frames with similarity to non-LTR retrotransposon proteins, including reverse transcriptase. A polymerase chain reaction assay was developed based on conserved amino acid sequences shared between the Ta11-1 reverse transcriptase and those of non-LTR retrotransposons from other species. Seventeen additional A. thaliana reverse transcriptases were identified that range in nucleotide similarity from 48-88% (Ta12-Ta28). Phylogenetic analyses indicated that the A. thaliana sequences are more closely related to each other than to elements from other organisms, consistent with the vertical evolution of these sequences over most of their evolutionary history. One sequence, Ta17, is located in the mitochondrial genome. The remaining are nuclear and of low copy number among 17 diverse A. thaliana ecotypes tested, suggesting that they are not highly active in transposition. The paucity of retrotransposons and the small genome size of A. thaliana support the hypothesis that most repetitive sequences have been lost from the genome and that mechanisms may exist to prevent amplification of extant element families.

Isolation of the Arabidopsis ABI3 gene by positional cloning.

Arabidopsis abi3 mutants are altered in various aspects of seed development and germination that reflect a decreased responsiveness to the hormone abscisic acid. The ABI3 gene has been isolated by positional cloning. A detailed restriction fragment length polymorphism (RFLP) map of the abi3 region was constructed. An RFLP marker closely linked to the abi3 locus was identified, and by analyzing an overlapping set of cosmid clones containing this marker, the abi3 locus was localized within a 35-kb region. An 11-kb subfragment was then shown to complement the mutant phenotype in transgenic plants, thereby further delimiting the position of the locus. A candidate ABI3 gene was identified within this fragment as being expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. In the most severe abi3-4 allele, the size of this predicted protein was reduced by 40% due to the presence of a point mutation that introduced a premature stop codon. The predicted ABI3 protein displays discrete regions of high similarity to the maize viviparous-1 protein.

Identification and map position of YAC clones comprising one-third of the Arabidopsis genome.

YAC clones corresponding to 125 Arabidopsis thaliana RFLP markers have been identified. At least one YAC clone has been isolated for each of the RFLP markers tested. Based on CHEF gel analysis of 196 clones, the mean insert size of the available Arabidopsis YAC libraries is approximately 160 kb. The YACs of known genetic map location encompass about 30% of the Arabidopsis genome. The results presented here represent a first step towards assembly of an overlapping YAC library of the A. thaliana genome.

Mapping the Arabidopsis genome.

We are engaged in a project to assemble a complete physical map of the Arabidopsis thaliana (L.) Heynh. genome. The first stage of this project involved the analysis of approximately 20,000 random cosmid clones representing an 8- to 10-fold sampling redundancy. Using computer matching programs, these clones have been assembled into some 750 contigs, encompassing 90-95% of the Arabidopsis genome. We are currently attempting to bridge the gaps by selecting the missing clones by hybridization. As a complement to this project we have constructed an RFLP map which currently contains 175 markers. The RFLP map provides contact points between the physical map and classical genetic map. Our main objective for undertaking this project is to simplify the cloning of genes where only the locus and not the product of the gene is known. In other words, the combined RFLP/physical map serves as a general cloning tool by simplifying the movement from the genetic locus to the cloned gene.

Restriction Fragment Length Polymorphism Linkage Map of Arabidopsis thaliana.

We have constructed a restriction fragment length polymorphism (RFLP) linkage map of the nuclear genome of the small flowering plant Arabidopsis thaliana. The map is based on the meiotic segregation of both RFLP and morphological genetic markers from five independent crosses. The morphological markers on each of the five chromosomes were included in the crosses to allow alignment of the RFLP map with the established genetic map. The map contains 94 new randomly distributed molecular markers (nine identified cloned Arabidopsis genes and 85 genomic cosmid clones) that detect polymorphisms between the Landsberg erecta and Columbia races. In addition, 17 markers from an independently constructed RFLP map of the Arabidopsis genome [Chang, C., Bowman, J.L., DeJohn, A.W., Lander, E.S., and Meyerowitz, E.M. (1988). Proc. Natl. Acad. Sci. USA 85, 6856-6860] have been included to permit integration of the two RFLP maps.

Effects of clindamycin and cefuroxime on leukocyte membrane receptors and function.

Normal human granulocytes and lymphocytes were preincubated in 0.5, 5, or 50 mg/l of clindamycin, and 5, 50, or 500 mg/l of cefuroxime. Incubation with clindamycin caused an increase in the proportion of granulocytes bearing receptors for the Fc portion of IgG (Fc gamma-R) and C3b (C3b-R). Random migration in capillary tubes was significantly decreased, but phagocytosis as measured by chemiluminescence was only slightly decreased. The proportion of lymphocytes bearing Fc gamma-R was decreased, while there was no effect on lymphocyte C3b-R percentage, nor on the proportion of granulocytes or lymphocytes bearing sheep erythrocyte (E) receptors. Preincubation of granulocytes in cefuroxime was not associated with changes in the proportion of receptor-bearing cells, except for a slight increase in Fc gamma-R-bearing lymphocytes at the lowest concentration tested. Tube migration was not affected but chemiluminescence was significantly decreased. Preincubation with clindamycin, although increasing the proportion of cells bearing phagocytosis-associated receptors, is thus not associated with an increased phagocytic ability, while cefuroxime incubation caused a decrease in chemiluminescence despite normal levels of receptor-positive cells.

A cautionary note on the use of blot hybridization for RNA size determination.

The ease with which RNA blot hybridization analysis can be performed makes it among the most widely used analytical tools in molecular biology. Hybridization with a labeled probe, subsequent to size fractionation and immobilization on a filter, allows one to approximate both the size and abundance of a given RNA in a single experiment. This communication demonstrates that dramatic differences in the electrophoretic mobility of the yeast GCN2 transcript are observed when size fractionation on formaldehyde gels is compared to fractionation of glyoxalated RNA. Both routinely used systems are considered to be fully denaturing. The anomalous mobilities therefore pose a potential problem when size determination is performed using a single gel system.

Transcriptional-translational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase.

GCN4 protein mediates the transcriptional activation of amino acid biosynthetic genes in Saccharomyces cerevisiae by specifically binding to DNA sequences in their 5'-regulatory regions. GCN4 expression is regulated at the level of translation, with translational derepression occurring under conditions of amino acid starvation. The product of the GCN2 gene is essential for translational derepression of GCN4. Sequence analysis of the GCN2 gene reveals that the GCN2 protein has a domain highly homologous to the catalytic domain of all known protein kinases. Furthermore, gcn2 strains are deficient in a protein kinase activity corresponding to a protein with the calculated molecular weight deduced from the GCN2 open reading frame. Therefore it is likely that GCN2 encodes a protein kinase, which may be directly involved in translational regulation of the GCN4 mRNA. Transcription of the GCN2 gene is increased when cells are cultured in amino acid starvation medium. This transcriptional activation is mediated by the GCN4 protein, which binds to the promoter region of the GCN2 gene. Thus, this system is modulated by a transcriptional-translational regulatory circuit, which is activated by amino acid starvation. Activation is not the result of a simple quantitative increase of either one of the identified components of the circuit.