Planning and implementation of a FIGO postpartum intrauterine device initiative in six countries.

OBJECTIVE: To describe the process of planning and implementing a program of counselling and delivery of postpartum intrauterine devices (PPIUD) in 48 hospitals across six countries in Africa and Asia. METHODS: The process of planning the FIGO PPIUD initiative, selection of countries and hospitals, model of implementation, and lessons for the future are described. RESULTS: Country-level and hospital-based leadership were essential and training-the-trainer models were successful. There was a need for consistency of competency standards allowing for national variations. As the project progressed, additional steps were necessary for steady implementation of the initiative, specifically: establishment of a project steering committee and a data safety monitoring committee, audits of structure and process, and regular feedback of each center's performance to stimulate maintenance and enhancement of activities. Postnatal follow-up was challenging in many countries with fragmented maternity systems. CONCLUSION: The importance of professional leadership and commitment backed by robust data for monitoring and feedback are essential for success.

Impact of contraceptive counselling training among counsellors participating in the FIGO postpartum intrauterine device initiative in Bangladesh.

OBJECTIVE: To evaluate the impact of structured training given to dedicated family planning counsellors on postpartum intrauterine device (PPIUD) services across six tertiary hospitals in Bangladesh. METHODS: Family planning counsellors underwent structured training on postpartum family planning, PPIUD in particular, over a four-day period. Impact of training was evaluated by comparing PPIUD counselling rates, consent rates, insertion rates, and removal rates five months before and five months after the training, using data from women delivering in the participating facilities. RESULTS: A total of 27 622 women were included in this analysis: 11 263 (40.8%) before the training intervention and 16 359 (59.2%) after it. There was an increase in the proportion of women who were counselled (from 75.3% to 83.8%, P<0.001), and a small decrease in the proportion of women agreeing to have a PPIUD inserted following counselling (13.7% vs 12.9%, P=0.03). Overall insertion rate was similar before and after training (9.5% vs 9.8%, P=0.42), while removal rate reduced from 2.8% to 1.8% (P=0.41). CONCLUSION: Structured training had no impact on overall PPIUD insertion rate. However, it did impact numbers of women receiving counselling, perceived quality of the counselling received, and overall removal rates.

FIGO postpartum intrauterine device initiative: Complication rates across six countries.

OBJECTIVE: To record and analyze complication rates following postpartum intrauterine device (PPIUD) insertion in 48 hospitals in six countries: Sri Lanka, India, Nepal, Bangladesh, Tanzania, and Kenya. METHODS: Healthcare providers were trained in counselling and insertion of PPIUD via a training-the-trainer model. Data were collected on methodology, timing, cadre of staff providing care, and number of insertions. Data on complications were collected at 6-week follow-up. Statistical analysis was performed to elucidate factors associated with increased expulsion and absence of threads. RESULTS: From May 2014 to September 2017, 36 766 PPIUDs were inserted: 53% vaginal and 47% at cesarean delivery; 74% were inserted by doctors. Follow-up was attended by 52%. Expulsion and removal rates were 2.5% and 3.6%, respectively. Threads were not visible in 29%. Expulsion was less likely after cesarean insertion (aOR 0.33; 95% CI, 0.26-0.41), following vaginal insertion at between 10 minutes and 48 hours (aOR 0.59; 95% CI, 0.42-0.83), and when insertion was performed by a nurse (aOR 0.33; 95% CI, 0.22-0.50). CONCLUSION: PPIUD has low complication rates and can be safely inserted by a variety of trained health staff. Given the immediate benefit of the one-stop approach, governments should urgently consider adopting this model.

Clinical outcomes of postpartum intrauterine devices inserted by midwives in Tanzania.

OBJECTIVE: To assess the rate of complications following immediate postpartum insertion of intrauterine devices (IUDs) by trained midwives in Tanzania. METHODS: A prospective cohort study of women who underwent immediate postpartum IUD (PPIUD) insertions provided by midwives between December 31, 2016 and October 15, 2017. Midwives received standardized training via the FIGO initiative. Women who returned 6 weeks after delivery were evaluated for complications. Outcomes of interest were uterine infection, IUD expulsion, medical removal of IUD, and method discontinuation. RESULTS: There were 40 470 deliveries, 2347 (5.8%) PPIUD insertions, and 1013 (43.2%) women with a PPIUD who returned for a follow-up visit in the program-affiliated clinics. Midwives were providers in 596 (58.8%) of these follow-up cases and clinicians in 417 (41.2%) cases. All PPIUD insertions by midwives were transvaginal and among them 43 (7.2%) had PPIUD-related complications by the end of sixth week. These complications included 16 (2.7%) cases of uterine infection, 14 (2.3%) IUD expulsions, 26 (4.4%) IUD removals, and 33 (5.5%) with overall method discontinuation. Only one case had uterine infection severe enough to warrant hospitalization. CONCLUSION: PPIUD insertion by trained midwives in Tanzania compares favorably with results reported from other settings.

Factors influencing the likelihood of acceptance of postpartum intrauterine devices across four countries: India, Nepal, Sri Lanka, and Tanzania.

OBJECTIVE: To examine the factors that positively influenced the likelihood of accepting provision of postpartum intrauterine devices (PPIUDs) across four countries: Sri Lanka, Nepal, Tanzania, and India. METHODS: Healthcare providers were trained across 24 facilities in counselling and insertion of PPIUDs as part of a large multicountry study. Women delivered were asked to take part in a 15-minute face-to-face structured interview conducted by in-country data collection officers prior to discharge. Univariate analysis was performed to investigate factors associated with acceptance. RESULTS: From January 2016 to November 2017, 6477 health providers were trained, 239 033 deliveries occurred, and 219 242 interviews were conducted. Of those interviewed, 68% were counselled on family planning and 56% on PPIUD, with 20% consenting to PPIUD. Multiple counselling sessions was the only factor resulting in higher consent rates (OR 1.30-1.39) across all countries. Odds ratios for women's age, parity, and cadre of provider counselling varied between countries. CONCLUSION: Consent for contraception, specifically PPIUD, is such a culturally specific topic and generalization across countries is not possible. When planning contraceptive policy changes, it is important to have an understanding of the sociocultural factors at play.

Sensitized genetic backgrounds reveal differential roles for EGF repeat xylosyltransferases in Drosophila Notch signaling.

In multicellular organisms, glycosylation regulates various developmental signaling pathways including the Notch pathway. One of the O-linked glycans added to epidermal growth factor-like (EGF) repeats in animal proteins including the Notch receptors is the xylose-xylose-glucose-O oligosaccharide. Drosophila glucoside xylosyltransferase (Gxylt) Shams negatively regulates Notch signaling in specific contexts. Since Shams adds the first xylose residue to O-glucose, its loss-of-function phenotype could be due to the loss of the first xylose, the second xylose or both. To examine the contribution of the second xylose residues to Drosophila Notch signaling, we have performed biochemical and genetic analysis on CG11388, which is the Drosophila homolog of human xyloside xylosyltransferase 1 (XXYLT1). Experiments in S2 cells indicated that similar to human XXYLT1, CG11388 can add the second xylose to xylose-glucose-O glycans. Flies lacking both copies of CG11388 (Xxylt) are viable and fertile and do not show gross phenotypes indicative of altered Notch signaling. However, genetic interaction experiments show that in sensitized genetic backgrounds with decreased or increased Notch pathway components, loss of Xxylt promotes Delta-mediated activation of Notch. Unexpectedly, we find that in such sensitized backgrounds, even loss of one copy of the fly Gxylt shams enhances Delta-mediated Notch activation. Taken together, these data indicate that while the first xylose plays a key role in tuning the Delta-mediated Notch signaling in Drosophila, the second xylose has a fine-tuning role only revealed in sensitized genetic backgrounds.

VH1 Family Immunoglobulin Repertoire Sequencing after Allogeneic Hematopoietic Stem Cell Transplantation.

After allogeneic hematopoietic stem cell transplantation (HSCT), recovery of humoral immunity is essential to protect from life-threatening infections. However, monitoring the humoral immune system after transplantation with standard techniques in the clinical routine is imprecise. Here, we performed sequencing of mononuclear bone marrow cells to characterize the VH1-repertoire of switched B cells of healthy volunteers and patients undergoing HSCT. Analysis of healthy bone marrow donors and patients showed virtually no clonally related sequences between individuals. Interestingly, clonally related sequences were present in pre- and post-transplantation bone marrow of patients undergoing HSCT for acute myeloid leukemia treatment. We consistently observed such related B cell clones, irrespective of conditioning regimen, donor source or time post transplantation. In general, repertoire diversity was lower in post-HSCT as compared to pre-HSCT samples. However, post-HSCT repertoires retained highly mutated sequences, despite immunosuppressive therapy and presence of T cell deficiency after HSCT. These observations identify key properties of the recovering B cell compartment and provide a conceptual framework for the surveillance of humoral immunity after allogeneic transplantation.

BALB/c and C57BL/6 Mice Differ in Polyreactive IgA Abundance, which Impacts the Generation of Antigen-Specific IgA and Microbiota Diversity.

The interrelationship between IgAs and microbiota diversity is still unclear. Here we show that BALB/c mice had higher abundance and diversity of IgAs than C57BL/6 mice and that this correlated with increased microbiota diversity. We show that polyreactive IgAs mediated the entrance of non-invasive bacteria to Peyer's patches, independently of CX3CR1(+) phagocytes. This allowed the induction of bacteria-specific IgA and the establishment of a positive feedback loop of IgA production. Cohousing of mice or fecal transplantation had little or no influence on IgA production and had only partial impact on microbiota composition. Germ-free BALB/c, but not C57BL/6, mice already had polyreactive IgAs that influenced microbiota diversity and selection after colonization. Together, these data suggest that genetic predisposition to produce polyreactive IgAs has a strong impact on the generation of antigen-specific IgAs and the selection and maintenance of microbiota diversity.

Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota.

Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis. However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how maternal antibodies are optimized throughout life to protect the newborn.

In vitro assays of orphan glycosyltransferases and their application to identify Notch xylosyltransferases.

Here we describe a systematic approach to determine the activity of putative glycosyltransferases with a focus on orphan members of the glycosyltransferase 8 family. An assay that measures the hydrolysis activity of glycoslytransferases can indicate the donor nucleotide sugar specificity without previous knowledge about the acceptor. Knowing the donor specificity, the acceptor specificity can subsequently be determined using synthetic acceptors. Three putative glycosyltransferases, now renamed GXYLT1, GXYLT2, and XXYLT1, have been identified this way as xylosyltransferases and in addition have been shown to act on O-glucosylated EGF repeats of Notch.

Negative regulation of notch signaling by xylose.

The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14-20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16-20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16-20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16-20 of Notch.

Site-specific O-glucosylation of the epidermal growth factor-like (EGF) repeats of notch: efficiency of glycosylation is affected by proper folding and amino acid sequence of individual EGF repeats.

O-Glucosylation of epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch is essential for Notch function. O-Glucose can be elongated by xylose to the trisaccharide, Xylα1-3Xylα1-3Glcß1-O-Ser, whose synthesis is catalyzed by the consecutive action of three glycosyltransferases. A UDP-glucose:protein O-glucosyltransferase (Poglut/Rumi) transfers O-glucose to serine within the O-glucose consensus. Subsequently, either of two UDP-xylose:glucoside xylosyltransferases (Gxylt1 or Gxylt2) transfers xylose to O-glucose. Finally, a UDP-xylose:xyloside xylosyltransferase (Xxylt1) transfers xylose to Xylα1-3Glcß1-O-EGF. Our prior site-mapping studies demonstrated that O-glucose consensus sites are modified at high but variable stoichiometries in mouse Notch1 and identified a novel glycosylation site with alanine in place of proline, suggesting a revised, broader consensus sequence (CXSX(P/A)C). Here we examined the molecular basis for this site specificity. A panel of EGF repeats from human coagulation factor 9 (FA9), mouse Notch1, and Notch2 were bacterially expressed and purified by reverse phase HPLC for use in in vitro enzyme assays. We demonstrate that proper folding of EGF repeats is essential for glycosylation by Poglut/Rumi, that alanine can substitute for proline in the context of coagulation factor 9 EGF repeat for O-glucose transfer, confirming the new consensus sequence, and that positively charged residues within the O-glucose consensus sequence reduce efficiency of glycosylation by Poglut/Rumi. Moreover, proper folding of EGF repeats is also important for the activities of Gxylt1, Gxylt2, and Xxylt1. These results indicate that protein folding and amino acid sequences of individual EGF repeats fundamentally affect both attachment and elongation of O-glucose glycans.

Molecular cloning of a xylosyltransferase that transfers the second xylose to O-glucosylated epidermal growth factor repeats of notch.

The extracellular domain of Notch contains epidermal growth factor (EGF) repeats that are extensively modified with different O-linked glycans. O-Fucosylation is essential for receptor function, and elongation with N-acetylglucosamine, catalyzed by members of the Fringe family, modulates Notch activity. Only recently, genes encoding enzymes involved in the O-glucosylation pathway have been cloned. In the Drosophila mutant rumi, characterized by a mutation in the protein O-glucosyltransferase, Notch signaling is impaired in a temperature-dependent manner, and a mouse knock-out leads to embryonic lethality. We have previously identified two human genes, GXYLT1 and GXYLT2, encoding glucoside xylosyltransferases responsible for the transfer of xylose to O-linked glucose. The identity of the enzyme further elongating the glycan to generate the final trisaccharide xylose-xylose-glucose, however, remained unknown. Here, we describe that the human gene C3ORF21 encodes a UDP-xylose:α-xyloside α1,3-xylosyltransferase, acting on xylose-α1,3-glucoseß1-containing acceptor structures. We have, therefore, renamed it XXYLT1 (xyloside xylosyltransferase 1). XXYLT1 cannot act on a synthetic acceptor containing an α-linked xylose alone, but requires the presence of the underlying glucose. Activity on Notch EGF repeats was proven by in vitro xylosylation of a mouse Notch1 fragment recombinantly produced in Sf9 insect cells, a bacterially expressed EGF repeat from mouse Notch2 modified in vitro by Rumi and Gxylt2 and in vivo by co-expression of the enzyme with the Notch1 fragment. The enzyme was shown to be a typical type II membrane-bound glycosyltransferase localized in the endoplasmic reticulum.

Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch epidermal growth factor repeats.

The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous alpha1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-d-xylose:beta-d-glucoside alpha1,3-d-xylosyltransferases adding the first xylose. The enzymes are specific for beta-glucose-terminating acceptors and UDP-xylose as donor substrate. Generation of the alpha1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose-deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells followed by mass spectrometric analysis of peptide fragments.