Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants

Deoxymugineic acid (DMA) is a member of the mugineic acid family phytosiderophores (MAs), which are natural metal chelators produced by graminaceous plants. Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe(III)–MAs complex. In contrast with barley, the roots of which secrete MAs in response to Zn deficiency, the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply. There was a concomitant increase in endogenous DMA in rice shoots, suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants. The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots. The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots; correspondingly, HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots. The expression of OsDMAS1 was increased in Zn-deficient shoots. Analyses using the positron-emitting tracer imaging system (PETIS) showed that Zn-deficient rice roots absorbed less 62Zn-DMA than 62Zn2+. Importantly, supply of 62Zn-DMA rather than 62Zn2+ increased the translocation of 62Zn into the leaves of Zn-deficient plants. This was especially evident in the discrimination center (DC). These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil.
Introduction
Zn, an essential element for plant growth, is absorbed from the rhizosphere by higher plants through specific transporters. The Zinc-regulated and Iron-regulated transporter Proteins (ZIP) have been isolated from both graminaceous (Ramesh et al. 2003; Ishimaru et al. 2005) and non-graminaceous plants (Grotz et al. 1998; Lasat et al. 2000; Moreau et al. 2002). Several ZIP transporters have been shown to be transcriptionally upregulated in Zn-deficient roots, but their contribution to the absorption of Zn from the rhizosphere into roots has not yet been demonstrated. Zn is also absorbed from the rhizosphere as complexes of Zn(II)–mugineic acid family phytosiderophores (MAs). The maize protein YS1 transports Fe(III)–MAs complexes (Curie et al. 2001), as well as other micronutrient-MAs complexes, including Zn(II)–MAs (Schaaf et al. 2004). von Wirén et al. (1996) reported that the ys1 mutant absorbed less 65Zn(II)-MAs than did the wild type, and Suzuki et al. (2006) showed that Zn-deficient barley absorbed more 62Zn(II)-DMA than 62Zn2+. These two studies also showed that free Zn2+ ions are absorbed into roots to some extent, suggesting that graminaceous plant roots absorb both Zn-DMA and Zn2+.
Graminaceous plants secrete MAs into the rhizosphere from their roots, although the amount differs among species in the order of: barley > wheat = rye > oat > maize > sorghum > rice (Takagi 1993). While the amount of MAs secreted is increased dramatically by Fe deficiency, it is not clear if Zn deficiency increases the secretion of MAs. Zn deficiency has been reported to increase (Cakmak et al. 1994; Walter et al. 1994; Zhang et al. 1989) or have no effect on the secretion of MAs from wheat and barley roots into the rhizosphere (Gries et al. 1995; Pedler et al. 2000).
The biosynthesis of MAs and their corresponding genes have been characterized (Fig. 3b) as being synthesized from methionine (Mori and Nishizawa 1987). Three molecules of S-adenosyl-l-methionine (SAM) are combined to form one molecule of nicotianamine (NA) by nicotianamine synthase (NAS). NA is then converted to the 3′′-keto acid by NA aminotransferase (NAAT), and 2′-deoxymugineic acid (DMA) is synthesized by DMA synthase (DMAS). In some graminaceous species, including barley, DMA is further hydroxylated by two dioxygenases, IDS2 and IDS3 (Kobayashi et al. 2001; Nakanishi et al. 2000). The genes encoding the enzymes involved in DMA synthesis have been well-characterized in Fe-deficient rice and barley. The expression of HvNAS1, HvNAAT-A, HvNAAT-B, HvDMAS1, HvIDS2, and HvIDS3 is increased in Fe-deficient barley roots (Nakanishi et al. 1993; Okumura et al. 1994; Higuchi et al. 1999; Takahashi et al. 1999; Bashir et al. 2006). In rice, the expression of OsNAS1, OsNAS2, OsNAAT1, and OsDMAS1 is increased in both roots and shoots by Fe deficiency (Inoue et al. 2003; Bashir et al. 2006). The expression of OsNAS3 is increased in Fe-deficient roots, but decreased in Fe-deficient shoots (Inoue et al. 2003). Promoter-GUS analysis suggests that OsNAS1 and OsNAS2 are involved in DMA secretion, because those genes are expressed in all root cells. On the other hand, OsNAS3 may not be involved in DMA secretion because its expression is restricted to the pericycle and companion cells of the roots (Inoue et al. 2003).
Recently, we showed that the expression of NASHOR2, a NAS gene in barley (Herbik et al. 1999), and HvNAAT-B was increased in Zn-deficient barley shoots, but that the expression of IDS2 and IDS3 was not detected in Zn-deficient shoots (Suzuki et al. 2006). On the other hand, the expression of HvNAS1, HvNAAT-A, HvNAAT-B, IDS2, and IDS3 was increased in both Zn- and Fe-deficient barley roots, while the expression of these genes was not evident in Fe-deficient shoots (Suzuki et al. 2006). This suggests that in barley Zn deficiency induces DMA synthesis in shoots, while both Zn and Fe deficiency induce MAs synthesis and secretion in roots.
The aims of the present study were to examine if the stimulation of MAs secretion by Zn deficiency is common in graminaceous plants and roles of MAs in Zn homeostasis of rice plants. In this report, we shows that DMA secretion is decreased in rice roots, while the concentration of endogenous DMA is increased in Zn-deficient rice shoots.
Materials and methods
Plant material and growth conditions
Rice seeds (Oryza sativa L. cv. Nipponbare) were germinated for 7 days at room temperature on paper towels soaked with distilled water. After germination, the seedlings were transferred to a Saran net floating on nutrient solution (Higuchi et al. 2001) and grown in a glasshouse. After 4 days, 270 seedlings were transferred to six 20-l plastic containers containing nutrient solution. After a further 5 days, 90 seedlings were transferred to two 20-l plastic containers containing nutrient solution without Zn followed 7 days later when a further 90 seedlings were transferred to two 20-l plastic containers containing nutrient solution without Fe. All samples were harvested 5 days later for analysis. Consequently, the Zn-deficient treatment was performed for 12 days, and Fe-deficient treatment for 5 days since Fe deficiency has a much more rapid deleterious effect. The nutrient solutions were renewed weekly with adjustment to pH 5.5 every 2 days with KOH.
Measurement of DMA secretion and endogenous levels of DMA and NA
DMA secretion was measured as described by Suzuki et al. (2006) and the concentrations of endogenous DMA and NA were measured as described by Higuchi et al. (2001). Briefly, the roots of 12 plants were rinsed with deionized water and then placed in 600 ml of deionized water at sunrise. Root exudates were collected for two periods of 2 and 3 h. After collection, Micropur (Katadyn, Switzerland), an antimicrobial agent, was added to prevent microbial degradation of the MAs Both solutions containing root exudates were combined and filtered (Advantec 5C, Toyo Roshi Kaisha, Ltd., Japan).
The shoots of the plants from which root exudates were collected were ground in liquid nitrogen using a mortar and pestle. Approximately 1 g of material was suspended in 40 ml of distilled water at 80°C for 30 min and filtered. The cationic fraction of the samples was prepared as a 2-M NH4OH eluate from Amberlite IR(H+)120 (Rohm and Haas Co., USA). The condensed and microfiltered samples were subjected to HPLC analysis, as described previously (Mori et al. 1987; Shojima et al. 1989). To ensure better separation of DMA, the pH of 0.15-N Li-citrate buffer was decreased to 2.97 and the pH of 0.2-N Li-citrate buffer increased to 3.30 for better separation of NA.
Northern blot analysis
The samples for Northern blot analysis were harvested 3 h after sunrise. The roots and shoots were separated immediately and stored at −80°C. Total RNA was extracted from the roots and shoots using SDS-phenol. Ten micrograms of total RNA were electrophoresed in 1.2% (w/v) agarose gels containing 0.66 M formaldehyde, then transferred to a Hybond-N+ membrane (Amersham, USA) and hybridized with probes at 65°C. The blots were analyzed using a Bio-imaging Analyzer System (BAS) 3000 (Fuji Film, Japan). The following specific primers were used to prepare specific probes: OsNAS1, 5′-GTCTAACAGCCGGACGATCGAAAGG-3′ and 5′-TTTCTCACTGTCATACACAGATGGC-3′; OsNAS2, 5′-TGAGTGCGTGCATAGTAATCCTGGC-3′ and 5′-CAGACGGTCACAAACACCTCTTGC-3′; OsNAS3, 5′-GACTGCTTCCATCGCTTGCTACCTC-3′ and 5′-CGCAACAGAGACAATGGTTGATTGT-3′; OsNAAT1, 5′-TAAGAGGATAATTGATTTGCTTAC-3′ and 5′-CTGATCATTCCAATCCTAGTACAAT-3′; and OsDMAS1, 5′-GCCGGCATCCCGCAGCGGAAGATCA-3′ and 5′-CTCTCTCTCTCGCACCTGCTAGCGT-3′. The accession numbers of the genes are AB021746 (OsNAS1), AB023818 (OsNAS2), AB023819 (OsNAS3), AB206814 (OsNAAT1), and AB269906 (OsDMAS1).
PETIS analysis of 62Zn translocation
PETIS experiment was established by Kume et al. (1997). 62Zn (half life: 9.13 h) was produced by the method of Watanabe et al. (2001). After adjustment of the 62Zn2+ solution to about pH 4 with 1 M KOH, 62Zn2+ was chelated with DMA in darkness for more than 3 h. Prior to the analysis, Zn-deficient rice plants were grown as described above but in a growth chamber (day: 30°C, 14 h of light at 320 μmol photons m−2 sec−1; night: 10 h at 25°C). The Zn-deficient rice plants were each supplied with 15 ml of culture solution lacking Zn in a polyethylene bag. The plants and bags were fixed between two acrylic boards and placed between a pair of PETIS detectors in a chamber set at 30°C, 65% humidity, and a light density of 320 μmol m−2 s−1. In the root absorption experiment, 62Zn (0.6 nmol) with DMA (79 nmol) was dissolved in 1 ml of distilled water and added to the culture solution. In the leaf absorption experiment, 62Zn (0.5 nmol) with DMA (33 nmol) was dissolved in 4.5 ml of nutrient solution containing cold Zn (2.5 μmol) and additional DMA (308 μmol), and supplied at the cut end of the second newest leaf. The individual leaves were cut to ensure a constant distance from discrimination center (DC) which is the junction of shoot and root (Kiyomiya et al. 2001). The γ-rays emitted from decaying positrons from 62Zn were counted over time using the coincident method with the paired detectors. The data were automatically corrected using 9.13 h as the half-life of 62Zn. The radioactivity of selected region was measured as region of interest (ROI) analysis. After PETIS analysis for 8 h, the plants were removed from the polyethylene bags, and the roots were gently washed for 1 min in 50 ml of 0.01 mM EDTA. Next, each plant was placed under a Bio-Imaging plate inside a cassette. The plate was scanned using an image analysis system (BAS-1500, Fuji Film, Japan). The plants were then cut into pieces, and the absolute amount of radioactivity was analyzed using γ-ray spectrometry with an ORTEC HPGe detector (Seiko EG & G Co., Ltd., Japan). Each experiment was conducted in triplicate. Due to the very short half-life of 62Zn, the radioactivity was different in each experiment. Therefore, images of individual plants and PETIS results are representative of those obtained.
Results
Plant growth, DMA synthesis and gene expression
In contrast with the good growth of the control rice plants, Zn or Fe deficiency had a markedly detrimental effect when plants were grown for 12 d or 5 d in solutions containing no Zn or Fe, respectively. Shoot growth was reduced by 50% in the absence of Zn and by 10% without Fe (Fig. 1). The corresponding figure for the Zn-deficient roots was a reduction of 20%, while the absence of Fe supply for 5 d had no significant effect on root growth. As previously established in our studies (Suzuki et al. 2006), there was no measurable effect of no Zn supply during the first week of growth (data not shown), but thereafter plants developed symptoms of Zn deficiency and growth declined. Chlorotic symptoms of Fe deficiency developed after 5 days of treatment while the fresh weights of the shoots and roots were almost the same as in the control plants.
Fig. 1Fresh weights of rice shoots and roots. 31-days old rice seedlings grown in hydroponically were harvested. Zn-deficient treatment (-Zn) was performed for 12 days, and Fe-deficient treatment (-Fe) was performed for 5 days. Values are mean ± SD (n = 4)
The amount of DMA secreted from the roots of Fe-deficient rice was much higher than that of the control plants (Fig. 2). On the other hand, Zn deficiency slightly decreased the level of DMA secretion. In fact, the amount of DMA secreted per plant in Zn-deficient rice was significantly decreased compared to that in the control plants (P < 0.05).
Fig. 2Amount of DMA secreted from rice roots. The growth condition and harvest time are the same in Fig. 1. Root exudates were collected for 5 h after sunrise. -Zn, zinc deficiency; -Fe, iron deficiency. Values are mean ± SD (n = 4)
We next analyzed the expression pattern of the genes participated in DMA synthesis in roots and shoots under Zn or Fe deficiency (Fig. 3a, b). As reported previously, the transcription of OsNAS1, OsNAS2, OsNAS3, OsNAAT1, and OsDMAS1 in roots was increased by Fe deficiency (Inoue et al. 2003, 2008; Bashir et al. 2006). On the other hand, Zn deficiency enhanced the transcription of OsNAS3 and OsNAAT1, but not that of OsNAS1, OsNAS2, and OsDMAS1. The expression of OsNAS2 in roots was slightly decreased by Zn deficiency. The expression patterns of OsNAS1 and OsNAS2 were well correlated with the amount of DMA secreted from the roots of Fe-deficient or Zn-deficient rice plants (Figs. 2, 3a).
Fig. 3Expression patterns of genes involved in DMA synthesis (a) Northern blot analysis of each gene involved in DMA synthesis. Each lane contained 10 μg of total RNA. C, control; -Zn, zinc deficiency; -Fe, iron deficiency. (b) DMA synthesis from S-adenosyl-l-methionine (SAM)
In the shoots, Fe deficiency increased the expression of OsNAS1, OsNAS2, OsNAAT1, and OsDMAS1, but decreased that of OsNAS3. In comparison, Zn deficiency increased the expression of OsNAS3 and especially that of OsNAAT1. The expression of OsDMAS1 was slightly increased in Zn-deficient shoots. Transcripts of OsNAS1 and OsNAS2 were not detected in control or Zn-deficient shoots (Fig. 3a).
Higuchi et al. (2001) reported that the concentration of endogenous DMA was increased in Fe-deficient rice shoots. We confirmed that the concentration of endogenous DMA in Fe-deficient shoots was about three times higher than that in control shoots (Fig. 4a). The concentration of endogenous DMA in Zn-deficient shoots was about two times higher than that in control shoots (Fig. 4b). Although the concentration of endogenous DMA in Zn-deficient shoots was lower than that in Fe-deficient shoots, the expression of OsNAAT1, which encodes the enzyme that converts nicotianamine to its 3′′-keto acid, in Zn-deficient shoots was much higher than that in Fe-deficient shoots (Fig. 3a). Therefore, we measured the endogenous NA, which is an intermediate of DMA. Corresponding to the expression pattern of OsNAAT1, endogenous NA in Zn-deficient shoots was under the detection limit by HPLC, while in Fe-deficient shoots NA was lower than in the control shoots (Fig. 4b).
Fig. 4Concentration of endogenous DMA and NA in rice shoots (a) Concentration of endogenous DMA. (b) Concentration of endogenous NA. The plants were harvested after collection of root exudates (Fig. 2), then DMA or NA was measured using HPLC. -Zn, zinc deficiency; -Fe, iron deficiency; N.D., not detected. Values are mean ± SD (n = 4)
Contribution of DMA to Zn translocation in Zn-deficient rice
To investigate whether DMA is involved in Zn absorption and translocation, a PETIS experiment was performed using Zn-deficient rice (Fig. 5; supplemental movie 1, supplemental Fig. 1). 62Zn chelated with DMA (62Zn-DMA) or 62Zn without DMA (62Zn2+) was supplied to Zn-deficient rice roots. Images of the plants and the localization of 62Zn after 6 h of absorption are shown in Fig. 5a. In both plants, most of the 62Zn absorbed into the plant was observed at the DC and in the bottom of the leaf sheath (Fig. 5a). The radioactivity of the plant supplied with 62Zn2+ was higher than that of the plant supplied with 62Zn-DMA both in the roots and shoots (Fig. 5b, c). This indicates that compared to Zn-DMA, Zn2+ is the preferred form for root absorption in Zn-deficient rice. Real-time imaging of the 62Zn absorption was observed by PETIS analysis (Fig. 5d). More 62Zn was observed in the bottom of the leaf sheath supplied with 62Zn2+ than in that supplied with 62Zn-DMA. This observation was quantified by ROI analysis (Fig. 5e) On the other hand, beginning at 90 min after the start of the experiment, more 62Zn was observed in the newest leaf of the plant supplied with 62Zn-DMA than in that supplied with 62Zn2+ (Fig. 5d, f). This indicates that Zn-DMA is preferred over Zn2+ for translocation within Zn-deficient rice plants. Continuous PETIS imaging is shown in supplemental movie 1. We performed three experiments of 62Zn absorption. Due to the very short half-life of 62Zn, however, the radioactivity was different in each experiment. Therefore, we showed the relative radioactivity of each experiment in supplemental Fig. 1. In each experiment, 62Zn2+ was more absorbed than 62Zn-DMA.
Fig. 562Zn movement from roots in Zn-deficient rice using PETIS (a) Plant image (left) and BAS image (right). (b) Radioactivity in roots after 6 h of absorption. (c) Radioactivity in shoots after 6 h of absorption. (d) Time course of the accumulation of radioactivity as determined by PETIS analysis. The data were scored every 3 min, and the images shown were taken at 90-min intervals for 6 h after supplying 62Zn through the roots. (e) Region of interest (ROI) analysis of 62Zn movement in the bottom of the sheath white boxed in (d). (f) ROI analysis in the newest leaf yellow boxed in (d)
We conducted an additional PETIS experiment in which Zn-DMA or Zn2+ was supplied to Zn-deficient rice at the cut second newest leaf (Fig. 6; supplemental movie 2, supplemental Fig. 2). Plant images and the localization of 62Zn after 6 h of absorption are shown in Fig. 6a. Most of the 62Zn absorbed from the second newest leaf remained in that leaf, but some 62Zn was translocated to the other leaves and roots as well as to the DC and leaf sheath. The level of radioactivity in the entire plant, except for the second newest leaf, was higher in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2+ (Fig. 6b), while that of the second newest leaf was not higher in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2+ (supplemental Fig. 3). PETIS analysis showed that more 62Zn appeared continuously in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2+. This suggests that 62Zn-DMA is translocated more rapidly than 62Zn2+ within the plant.
Fig. 662Zn movement from cut leaf in Zn-deficient rice using PETIS (a) Plant image (left) and BAS image (right). Arrows indicate the point where 62Zn was absorbed. (b) Radioactivity in the entire plant except for the second newest leaf after 8 h of absorption. (c) Time course of the accumulation of radioactivity as determined by PETIS analysis. The data were scored every 3 min, and the images shown were taken at 120-min intervals for 8 h after supplying 62Zn through the shoots
Discussion
The concentration of endogenous DMA is increased in Zn-deficient shoots, but DMA secretion from the roots is not increased
Previously we reported increased MAs secretion in Zn-deficient barley (Suzuki et al. 2006); however, in rice, Zn deficiency did not increase the secretion of DMA, but rather slightly decreased it (Fig. 2). Fe deficiency increased MAs secretion in both rice and barley (Suzuki et al. 2006; Fig. 2). In barley, expression of the genes encoding the MAs biosynthetic enzymes was increased in the roots of both Zn- and Fe-deficient plants (Suzuki et al. 2006). On the other hand, in rice, the expression pattern of the genes involved in DMA synthesis in roots was increased in Fe-deficient roots, while the expression of OsNAS1, OsNAS2, and OsDMAS1 was not increased in Zn-deficient roots (Fig. 3a). Inoue et al. (2003) suggested that OsNAS1 and OsNAS2 are involved in DMA secretion. Supporting this idea, the expression pattern of OsNAS1 and OsNAS2 in roots was in good correlation with the amount of DMA secreted (Figs. 2, 3a). On the other hand, OsNAS3 is thought not to be involved in DMA secretion, because the expression of OsNAS3 is restricted to the pericycle cells adjacent to the protoxylem and companion cells but not in other cortical cells (Inoue et al. 2003). The expression of OsNAS3 is observed in whole leaf tissues in the control plants (Inoue et al. 2003). The expression of OsNAS3 was increased in Zn-deficient roots and shoots (Fig. 3a); therefore, OsNAS3 may function to synthesize NA or subsequent components, such as DMA, for the distribution of Zn within Zn-deficient plants. It is noteworthy that Zn deficiency enhanced the expression of OsNAS3 in shoots, while Fe deficiency enhanced the expression of OsNAS1 and OsNAS2. This suggests that NA and DMA are involved both in Fe and Zn homeostasis, but that their synthesis under conditions of Fe and Zn deficiency is catalyzed by different isozymes.
The concentration of endogenous DMA in the shoots was increased by Zn and Fe deficiencies (Fig. 4a). Although the induction of OsNAAT1 in Zn-deficient shoots was much higher than that in Fe-deficient shoots, the concentration of DMA in Zn-deficient shoots was lower than that in Fe-deficient shoots. In accordance with the high level of OsNAAT1 expression in Zn-deficient shoots, the concentration of NA in Zn-deficient shoots was low (Fig. 4b). This result led to us hypothesize that OsDMAS1 is a rate-limiting enzyme for DMA synthesis in Zn-deficient rice.
Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice
Recently, we showed that Zn-deficient barley absorbs more Zn-DMA than Zn2+ from its roots (Suzuki et al. 2006). In the present paper, however, less Zn-DMA than Zn2+ was absorbed from the roots of Zn-deficient rice (Fig. 5). These findings are correlated with the change in the amount of MAs secreted under Zn deficiency. In barley, MAs secretion is increased by Zn-deficiency, and this contributes to the absorption of Zn from the soil. On the other hand, in rice, the level of DMA secretion was slightly decreased (Fig. 2). This suggested that rice may prefer to absorb Zn2+ rather than Zn-DMA.
Our PETIS experiment clearly showed that more 62Zn was translocated to the newest leaf when 62Zn-DMA was supplied than when 62Zn2+ was supplied, while the opposite tendency was observed at the bottom of the leaf sheath (Fig. 5, supplemental movie 1). This finding suggests that Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice. The preferential translocation of 62Zn-DMA was also observed in the PETIS experiment in which 62Zn-DMA and 62Zn2+ were supplied from the Zn-deficient leaf (Fig. 6, supplemental movie 2). These data also support the idea that Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice.
Since DMA synthesis might be promoted by Zn deficiency in barley shoots (Suzuki et al. 2006), graminaceous plants have a common system for Zn translocation within the plant, while the contribution of MAs to Zn uptake from the soil differs among species. Further studies will enable us to understand the mechanism of Zn transport, to manipulate the distribution of Zn in order to produce crops that tolerate Zn-deficient stress, and to enhance the Zn content of crop seeds.
Electronic supplementary material
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deoxymugineic acid
rice
nicotianamine
transport
zinc
iron


Rituximab therapy for juvenile-onset systemic lupus erythematosus

Rituximab (RTX), an anti-CD20 monoclonal antibody, has been proposed for use in the therapy of systemic lupus erythematosus (SLE). We present the initial long-term experience of the safety and efficacy of rituximab for treatment of SLE in children. Eighteen patients (mean age 14 ± 3 years) with severe SLE were treated with rituximab after demonstrating resistance or toxicity to conventional regimens. There was a predominance of female (16/18) and ethnic African (13/18) patients. All had lupus nephritis [World Health Organization (WHO) classes 3–5] and systemic manifestations of vasculitis. Clinical disease activity of the SLE was scored with the SLE-disease activity index 2K (SLEDAI-2K). Patients were followed-up for an average of 3.0 ± 1.3 years (range 0.5 to 4.8 years). B-cell depletion occurred within 2 weeks in all patients and persisted for up to 1 year in some. Clinical activity scores, double-stranded DNA (dsDNA) antibodies, renal function and proteinuria [urine protein to creatinine ratio (Upr/cr)] improved in 93% of the patients. Five patients required multiple courses of RTX for relapse, with B-cell repopulation. One died of infectious endocarditis related to severe immunosuppression. In conclusion, our data support the efficacy of rituximab as adjunctive treatment for SLE in children. Although rituximab was well tolerated by the majority of patients, randomized controlled trials are required to establish its long-term safety and efficacy.
Introduction
Systemic lupus erythematosus (SLE) in children is known to carry a worse prognosis than in adults, especially in those of African and Hispanic ethnicity [1, 2]. Although treatment protocols for proliferative lupus nephritis have evolved during the past two decades, pediatric regimens have been center based and selectively derived from adult protocols [3]. These protocols primarily include cyclophosphamide (CYC), which carries long-term toxicities related to malignancy and gonadal dysfunction [4].
Most recently, B-lymphocyte function has been recognized as a major component in the pathogenesis of autoimmune diseases [5]. Rituximab (RTX), a chimeric monoclonal antibody directed against CD20 lymphocytes (commonly called B-cells), was developed as a primary treatment against B-cell lymphoma [6] and is now approved for use in the treatment of rheumatoid arthritis [7]. Although clinical trials of the use of RTX in adults with SLE are substantial and ongoing [8], studies in children have been limited [9, 10]. A collaborative retrospective and uncontrolled multicenter experience from Europe reported mixed results [11]. Unfortunately, that study failed to use a single protocol, and many patients were treated concurrently with more toxic medications, which made safety analysis unreliable [11].
The purpose of our study was to determine the safety and efficacy of RTX therapy in the treatment of children with severe SLE. Over the course of 7 years, we retrospectively assessed the indications and responses to RTX therapy in a cohort of children with active SLE that was refractory to conventional therapies or who had clinical indications prohibiting the use of more toxic immunosuppressant drugs.
Patients and methods
A retrospective analysis was performed on a cohort of 51 patients diagnosed with SLE and lupus nephritis who received their care at Holtz Children’s Hospital, University of Miami Miller School of Medicine, between January 1996 and June 2007. The study was approved by the institutional review board, with waiver of consent authorization, and all subjects were assured anonymity in compliance with the Health Insurance Portability and Accountability Act (HIPAA). The medical records were reviewed for patients’ demographic characteristics, age at diagnosis, prior and current medical treatments, type of lupus nephritis and serologic and clinical response to therapy.
Children were considered eligible for inclusion in the analysis if they fulfilled the following criteria: (1) American College of Rheumatology (ACR) criteria for the diagnosis of SLE [12]; (2) age < 16 years at the time of diagnosis of SLE; (3) treatment with the anti-CD20 monoclonal antibody, rituximab, during the observation period from January 2000 through October 2007.
Clinical protocol
Rituximab (RTX) was administered weekly for two to four doses. The initial dose was 188 mg/m2, infused over 4 h. Subsequent doses were 375 mg/m2 per dose, infused over 6 h to 8 h. Premedication initially consisted of diphenhydramine and acetaminophen, administered 30 min before the rituximab infusion. However, mild infusion reactions, including pruritis, nausea and emesis, resulted in our adding hydrocortisone 50–100 mg intravenously 30 min prior to each infusion. Lymphocyte subsets were assayed before and after each course of RTX therapy at 1- to 3-month intervals. This included B cells assayed as CD19+ lymphocytes and T-cells assayed as CD4 and CD8 lymphocytes as total and percent of the total lymphocyte cell count. The serum immunoglobulin levels were measured every 1 to 6 months prior to and after the course of RTX. Adverse events were recorded from the time of the first infusion until 1 year following the treatment course.
Lupus disease activity index and serology
Clinical and biological parameters were assessed prior to the RTX infusion course, at 1- to 3-month intervals thereafter. The clinical disease activity was scored with the SLE-disease activity index 2K (SLEDAI-2K), which has a maximum activity score of 105 [13]. Laboratory evaluations included serum complement components (C3 and C4) and anti-double stranded DNA (Anti-dsDNA) antibody and anti-nuclear antibody (ANA).
Lupus nephritis and renal disease activity
Renal biopsies were classified according to the new proposed modifications of the World Health Organization (WHO) classifications [14]. Three patients were treated at the time they were on hemodialysis for extra-renal manifestations of SLE. For those patients not on dialysis, degree of proteinuria and renal function were assessed in response to RTX therapy. Degree of proteinuria was determined by the random urine protein to creatinine ratio (Upr/cr) in milligrams per milligram (mg/mg), with normal <0.2 and nephrotic range >1.0 [15, 16]. Serum albumin (Salb) was assayed as an indirect measure of the nephrotic syndrome. Serum creatinine (Scr) was assayed, and an estimation of glomerular filtration rate (eGFR) was derived by the height index formula of Schwartz, expressed as milliliters per minute per 1.73 meters squared (ml/min per 1.73 m2) [17]. Complete renal remission was defined as normalization of Upr/cr and eGFR. Partial remission was defined as > 50% decline in Upr/cr. All serum and urine chemical analyses and immunoassays were performed in the central hospital clinical laboratory or the Quest referral laboratories.
Statistical methods
All data sets were analyzed for Gaussian distribution using the D’Agostino–Pearson omnibus test for normality. The data were calculated into average values at baseline and at 2 months, 4 months, and 6 months after initiation of treatment. Variables at baseline, during and after the treatment were compared by the Wilcoxon matched-pairs signed-ranks test. Differences in parameters between the 2 independent groups were analyzed using Mann-Whitney and the Friedman test with Dunn’s multiple comparisons post tests for repeated measures of nonparametric data. P values of less than 0.05 were considered significant. All results are expressed as mean ± standard deviation (SD).
Results
Patients’ demographics
During the period of observation 18 patients were treated with RTX for at least one course of two to four doses. There were 16 female patients and two male patients. Mean age at the time of diagnosis of SLE was 10.7 ± 2.5 years (range 7 to 14 years). Mean duration of disease prior to treatment with RTX was 3.1 ± 2.5 years (range 2 months to 8 years). Age at the time of first RTX treatment was 14.2 ± 3.3 years, and current age at the time of this report was 17 ± 5.0 years.
All patients had clinical evidence of lupus nephritis. Individual patient demographics, clinical manifestations of SLE and indications for rituximab therapy for patients not on hemodialysis are shown in Table 1. All patients received concurrent therapy with low-dose corticosteroids (CSs) and hydroxychloroquine (HCQ). Sixteen patients had failed or had suffered toxic effects from multiple courses of intravenous therapy with cyclophosphamide (CYC) and corticosteroids. Treatment with RTX included concurrent or subsequent treatment with maintenance doses of mycophenolate mofetil (MMF) or azathioprine (AZT) similar to recently published adult protocols [18].
Table 1Patients’ baseline characteristics, indications for rituximab treatment, and clinical course (F female, M male, H Hispanic, B Black, HT hypertension, CYC cyclophosphamide, CyA cyclosporine A, AZT azathioprine, MMF mycophenolate mofetil, IVIG intravenous therapy with gamma globulin, CS corticosteroids, HXQ hydroxychloroquine, FR full remission, PR partial remission)PatientGenderAge at diagnosis (years)RaceIndication for rituximabNephritis classPrevious and/or concurrent therapiesaUpr/cr (mg/mg)OutcomeBefore RTXAfter RTX1F12.0BNephritis, serositis, Coomb’s (+) anemia, CYC-toxicityIVCYC, MMF13.00.1FR2M10.0WNephritis, serositis, CS toxicityIVCYC, MMF2.10.6PR3F11.0BNephritis, serositis, Coomb’s(+) anemia, CYC- toxicity sepsisIVCYC, MMF4.01.7PR4F8.0HPulmonary HT, nephritis, Coomb’s(+) anemiaIV-VCYC, MMF, IVIG2.60.2FR5F12.0BOphthalmic vasculitis, nephritisIV-VCYC, AZT0.40.1FR6F14.0BNephritis, Coomb’s(+) anemia, CS toxicityNACYC, AZT, MMF, IVIG0.60.3PR7F12.0BNephritis, serositis, CYC-toxicity sepsisIV-VCYC, AZT, MMF, IVIG2.6NADied8F12.0BNephritis, serositis, Coomb’s(+) anemiaIVCYC, AZT, IVIG3.61.9PR9F14.0BNephritis, serositis, ataxia, miliary tuberculosisIIIIVIG1.70.2FR10F7.0HNephritis, serositis, arthritis, CYC-toxicity sepsisIVCYC, MMF, IVIG3.00.7PR11F12.8HNephritis, serositis, arthritis, obesityVMMF9.70.2FR12F7.0BNephritis, thrombocytopenia, CYC toxicityIV-VCYC, AZT, MMF, IVIG, CyA5.40.2FR13F7.0BNephritis, Coomb’s(+) anemia, skin vasculitisIVMMF1.50.2FR14M12.5HNephritis, serositis, arthritis, CYC-toxicity sepsisIIICYC, MMF, IVIG2.30.8PR15F13.0BNephritis, arthritis, skin vasculitis, Coomb’s(+) anemiaIVCYC, MMF, IVIG6.80.9PRaAll patients were treated with CS and HXQ
All three patients on hemodialysis responded positively to the RTX therapy. Patient HD-1 had Raynaud’s syndrome and finger necrosis; her symptoms resolved, and she recently received a transplant, 4 years after RTX treatment. Patient HD-2, who had been on ventilator support with pneumonitis, pleuritis and carditis for 1 month prior to receiving RTX, recovered and has been stable on hemodialysis as an outpatient for >4 years since RTX therapy. Patient HD-3, after 3 years on hemodialysis, developed debilitating chorea with increased lupus serology. She did not respond to anti-convulsant therapy. The chorea resolved within weeks of the RTX therapy. She remains on dialysis without recurrence of chorea.
B-cell depletion and re-population
Figure 1 shows the average B-cell depletion relative to the beginning of the first dose of RTX. Patients received an average of 4.3 ± 1.6 doses overall. B-cell depletion was achieved within 2 weeks of the first dose of RTX and lasted an average of 3 ± 4 months (range 3 to 12 months). Five patients (28%) received repeated courses of RTX therapy ranging from two to four courses of one to four additional doses following re-population of the B-cell line in the setting of clinical relapse of SLE. Overall follow-up period from the initial dose of RTX to this report was 3.0 ± 1.3 years. It should be noted that those patients who did not have clinical relapse were not routinely assayed for lymphocyte subsets. Therefore, it is not known if or when those patients with persistent clinical remission had their B-lymphocyte lineage re-populated.
Fig. 1Graph of B-cell depletion over time from before rituximab therapy to 12–18 months after rituximab therapy. Asterisks indicate significant difference from the post-rituximab values; P < 0.01
Table 2 describes the changes in the lymphocyte subsets from baseline to the nadir drop in the B-lymphocyte lineage. Total lymphocyte counts, as well as CD4 and CD8 T-cell counts, tended to be lower before RTX treatment and increased after RTX therapy. Similarly, the levels of serum immunoglobulins remained normal in comparison with the pre-RTX values.
Table 2Clinical parameters before and after rituximab therapy (NS not significant)ParameterBefore rituximab therapyAfter rituximab therapyPTotal lymphocyte count1044 ± 5961734 ± 1535NSCD4+ lymphocytes344 ± 217556 ± 264NSCD19+ lymphocytes243 ± 22374 ± 710.005Immunoglobulin IgG1603 ± 8481596 ± 1055NSImmunoglobulin IgM273 ± 514283 ± 531NSUpr/cr (mg/mg)4.0 ± 3.50.6 ± 0.60.001Serum albumin (g/dl)2.6 ± 0.73.5 ± 0.60.001Scr (mg/dl)1.2 ± 0.40.6 ± 0.20.001eGFR (ml/min/1.73m2)86 ± 32144 ± 370.0003Corticosteroid dose (mg/m2 per day)79 ± 2613 ± 20<0.0001SLEDAI-2K score47 ± 1925 ± 140.0004
Lupus activity and auto-antibody response
Table 2 and Fig. 2 summarize the clinical response to RTX therapy. The average lupus activity score (SLEDAI) improved in all patients. Average dsDNA antibody decreased and serum C3 complement increased significantly after RTX therapy. Other serological parameters showed a trend towards improvement as well.
Fig. 2Composite graphs depicting response to rituximab therapy on clinical parameters. Panela SLEDAI [13] scores before and after rituximab therapy. Panelb proteinuria (Upr/cr) before and after rituximab. Panelc auto-antibody titers before and after rituximab. Paneld serum complements as C3 and C4 complements. Asterisks indicate significant differences from the post-rituximab values; P < 0.01
All 15 patients not on dialysis had active lupus nephritis with significant proteinuria. Table 1 provides detailed information regarding the classification of the lupus nephritis for each patient, together with extra-renal manifestations and concurrent or previous therapies, as well as their final outcome. All parameters improved or stabilized in 14/15 (93%) patients. Seven had total remission of proteinuria, while the other seven had partial remission, with significant improvement in proteinuria and renal function. Coincident with the improvement in renal disease, the requirement for maintenance corticosteroid therapy was reduced in most patients, with an average fall in corticosteroid dosing from 79 ± 29 mg/m2 per day before RTX therapy to 13 ± 20 mg/m2 per day after RTX therapy (P < 0.0001).
Adverse events
One patient (#7) received three doses of RTX shortly after a cycle of intravenous cyclophosphamide (CYC) administration in an effort to control lupus serositis and nephritis. She developed Staphylococcus aureus endocarditis, with vegetations on the mitral and aortic valves. She died after open heart surgery. This was considered a death from overwhelming infection most likely related to excessive immune suppression. Her CD4 count prior to RTX was 220/mm3, possibly related to prior CYC therapy. At the time of her death her serum immunoglobulin levels were normal.
Patient #13 had systemic vasculitis and class IV lupus nephritis. Since she was only 7 years of age, it was decided to treat her with intravenous administration of Solu-Medrol followed by weekly RTX for four doses and concurrent mycophenolate mofetil (MMF) as induction therapy. She responded well to RTX, with resolution of her proteinuria and clinical remission of the lupus nephritis. However, she developed symptoms of cerebral vasculitis, with seizures and a cerebral infarct, demonstrated by magnetic resonance imaging (MRI) after her fourth dose of RTX. She was treated with intravenously administered gamma globulin (IVIG) and two doses of cyclophosphamide. Her neurologic deficits resolved and her vasculitis reversed, as demonstrated by MRI. She remains in remission of the lupus nephritis and cerebritis 6 months after RTX therapy.
Mild adverse reactions occurred in approximately half the patients and included pruritis, nausea, or malaise associated with the infusions. These became less when hydrocortisone was added to the pre-medication regimen and the infusion rate was slowed to 6 to 8 hours.
Discussion
To date, this report has provided the largest long-term experience in children treated with targeted B-cell depletion using the monoclonal antibody, rituximab, for severe SLE. We showed a beneficial effect of the drug in over 90% of the children in this series, which is consistent with that of previous reports [8, 19–21]. Moreover, we were able to demonstrate efficacy in controlling proteinuria in conjunction with a fall in auto-antibodies in those patients with active lupus nephritis, which mirrored that in a series of adult patients [19].
B-lymphocyte dysregulation is central in the pathogenesis of systemic lupus, with disruption of an individual’s immune homeostasis. These mechanisms are multi-dimensional and are not limited to the single role of B cells in antibody production [22]. Hence, the elimination of the B-cell lineage, with potential re-population with a “healthier” line, is theoretically curative to the patient.
The safety of rituximab in young lupus patients remains unanswered. Most of the patients in our cohort tolerated the rituximab well. However, as in the European trial [11], our patients were on variable multi-drug regimens. The one patient who died did so from infectious complications in conjunction with severe immune suppression from multiple cytotoxic agents, including both rituximab and cyclophosphamide. Another young patient developed symptoms of cerebral vasculitis, with seizures and encephalopathy. The differential diagnosis included the dreaded progressive multifocal leukoencephalopathy (PML), recently reported to occur in patients treated with rituximab [23]. Although she showed clinical recovery after cyclophosphamide therapy, the case emphasizes the uncharted risks imposed by the use of drugs without well-studied indications.
The efficacy of rituximab may be short-lived in some patients. Resistance to RTX B-cell depletion may be related to antibodies against the human-mouse chimeric protein [8]. It may also be genetically influenced by polymorphisms of the FcRIIIa gene, responsible for the Fc-γ receptor required for RTX binding to phagocytes [24]. In our series, all patients had some degree of B-cell depletion after the initial course of RTX. However, five of the 18 patients (28%) developed clinical relapse in association with re-population of the B-cell lineage. All but one of these responded to additional courses of RTX. This is similar to recent long-term reports in a series of adults [25].
Treatment regimens in children with SLE do not conform with those of adults developed by carefully designed randomized controlled trials. This may be partially attributable to the fact that children ≤ 16 years of age are excluded from these trials. This has resulted in an ambiguous and often anecdotal approach to pediatric patients, especially those with early onset and severe disease. Although intravenous cyclophosphamide treatment was established as induction therapy for proliferative lupus nephritis over two decades ago [26], its short- and long-term toxicity delayed its incorporation into pediatric regimens [27]. Paradoxically, those with severe disease have received excessive courses of toxic medications in futile efforts to preserve renal function [27]. More recently, less toxic alternative treatment regimens, including mycophenolate mofetil for both induction and maintenance therapy, have resulted in equal or improved renal survival over conventional therapies [18, 28]. Since treatment protocols of children do not conform to conventional protocols for adults, attempts at reporting multicenter experiences are hampered by a lack of consistency in treatment regimens [11, 22]. Therefore, anecdotal center experiences such as our own are necessary in developing treatment protocols tailored towards efficacy while decreasing long-term risks on growth, fertility and malignancy.
In conclusion, RTX therapy offers a potential innovation in the current treatment regimens for children with aggressive SLE that includes both renal and extra-renal manifestations. The development of a randomized controlled treatment protocol for induction and maintenance therapy in pediatric patients is very much warranted.

rituximab
systemic lupus erythematosus
children


A Perspective of Preconception Health Activities in the United States

Objectives: Information regarding the type and scope of preconception care programs in the United States is scant. We evaluated State Title V measurement and indicator data and abstracts presented at the National Summit on Preconception Care (June 2005) in order to identify existing programs and innovative strategies for preconception health promotion.
Introduction
The June 2005 National Summit on Preconception Care, sponsored by the Centers for Disease Control and Prevention (CDC) and the March of Dimes, assembled an array of health care providers, public health practitioners, and researchers in an effort to promote the exchange of information and ideas related to preconception care at the local, state, and national levels. A key issue emerging from conference discussions was the perceived lack of information pertaining to the nature and scope of preconception health services currently being provided in the United States, whether explicitly defined or falling under another umbrella of services. Another issue was the recognition that a compilation of local, state, and national resources would provide important information related to the ability of current public health systems to address preconception care needs, as well as allow for the description of innovative models for the delivery of such services.
Attended by more than 400 registered participants representing a myriad of agencies and organizations, the summit generated significant response within the public health and clinical communities and reflects a perspective that cannot be readily gleaned from literature reviews or the inspection of existing programmatic guidelines: that is, the topic of preconception health has garnered substantial interest and support and has already been incorporated into numerous existing programs, policies, and strategies in the United States. Unique to the summit was an opportunity to learn about the development and administration of preconception care programs within clinical and population-based settings as Medline, PubMed, and other Internet searches have revealed very little information regarding the development and implementation of preconception care programs within a local context. However, as evidenced by the numerous programmatic components featured in the conference abstracts, such programs are being introduced in many different formats, among various private and public organizations, and across the entire nation.
Although little is now known at this writing about the scope and extent of U.S. programs and projects to promote preconception health, this article uses readily available sources (in the published professional literature and the gray literature of government and organization reports) to provide a snapshot of related activities in the United States. Thus, we are able to report the findings of a scan of State Title V Performance Measures and Priority Needs pertaining to preconception care and a detailed summary of the abstracts of the papers presented at the Summit.Fig. 1State Title V priority needs focused on preconception health and health care, U.S. 2005
Methods
The Maternal and Child Health Bureau website (http://mchb.hrsa.gov/) was used to ascertain the proportion of states including preconception health indicators in State Performance Measures and Priority Needs, as reported in the State Title V Maternal and Child Health Service (MCH) Block Grant FY 2004 Annual Report and FY 2006 Application, Form 14. The web-based Title V Information System (https://perfdata.hrsa.gov/mchb/mchreports/Search/core/ measureindicatemenu.asp) was used to search State Title V Performance Measures and Priority Needs for indicators related to preconception health. Initial search parameters included all keywords and all populations and were further refined by selecting for birth outcomes, reproductive health, and women. Those indicators for which infants, newborns, children, men, or service providers represented the sole target population were excluded. The remaining measures were evaluated based on their relevancy to preconception health. Indicators using the term “preconception” or “preconceptual” and those that did not use such terminology but adequately captured the concept were considered to fall within the domain of preconception health and health care. States identifying preconception health Priority Needs were depicted on a national map. States reporting relevant Performance Measures were identified and selected measures were described in tabular format. Finally, a comprehensive list of State Title V Performance Measures and Priority Needs related to preconception health, that reflected the tone and language of multiple states was developed.
All abstracts submitted and accepted for presentation at the National Summit on Preconception Care (June 2005) were pooled and subjectively categorized into five broad topic areas: research pertaining to preconception risk assessment and outcome evaluation; preconception care programs and activities; tools intended for provider or patient preconception health education; clinical practice strategies; and public policy related to preconception care.
Results
State Title V Performance Measures and Priority Needs
The federal MCH Block Grant guidance requires states to report annually on National “Core” Performance Measures and to assess needs and set priorities every 5 years. In addition, each state must develop 7 to 10 additional Performance Measures relevant to its own priority needs and programs. Priority Needs reflect a state’s focus for programmatic efforts throughout the next five years and can be adjusted during interim years in response to variations in needs. Performance Measures describe a specific maternal and child need that, when effectively addressed, can lead to improvements in health. In 2005, states completed the 5-year needs assessments and set out priorities and targets for 2010. A previous analysis of the 2000 priorities and measures found that some states, but not all, included measures for preconception care or visits, as did Healthy People 2000 National Health Promotion and Disease Prevention Objectives [1].
Twenty-three states reported a Priority Need that focused on preconception health and health care (Fig. 1). “Preconception” or “preconceptual” health was mentioned by 10 states as a Priority Need in maternal and child health. In other states, Priority Needs indirectly addressed preconception care via broad objectives such as improving the health of women of childbearing age, promoting reproductive health and sexual responsibility, and advancing a holistic care continuum for women’s health.
A total of 42 states and jurisdictions reported at least one Performance Measure related to preconception care, neural tube defects, birth spacing, family planning, unintended pregnancy, and healthy weight/obesity (Table 1). Unintended pregnancy and healthy weight or obesity were the measures identified most frequently (43% and 19%, respectively). The majority of the measures pertaining to birth spacing or parity, or both, addressed repeat pregnancies among adolescent populations. A total of thirty states or territories reported measures on tobacco use during pregnancy; however, only one focused on the proportion of women who smoked during 3 months prior to pregnancy. Measures pertaining to smoking rates among women of childbearing age were reported by two states. Four states addressed mental health among women of childbearing age and postpartum depression was identified in three others. Two states included measures pertaining to preventive oral health services among women of childbearing age (data not shown).
Table 2 provides examples of selected State Performance Measures and Priority Needs derived from 18 states. Variations in state-selected preconception health indicators are evident, with some states detailing various components of preconception care and others employing a broader focus on reproductive health.
Table 1States or U.S. jurisdictions reporting Performance Measures related to selected preconception health topicsPerformance measureStatePreconception careNeural tube defects/folic acidBirth spacing/parityFamily planningUnintended pregnancyHealthy weight/obesityArizona×Arkansas×California×Delaware×District of Columbia×a×Florida×aGeorgia××aGuam×Hawaii×Illinois×Indiana×Kentucky×Louisiana×Maine×Marshall Islands×bMaryland×Massachusetts××Michigan×Minnesota×Mississippi×aMontana×Nebraska×Nevada×New Jersey×aNew Mexico××New York×North Carolina×××North Dakota×Northern Mariana Islands×Ohio×Oklahoma×Puerto Rico×South Carolina×TennesseeTexas×South Dakota×Wyoming×cUtah×Vermont×Washington×bWisconsin×aMeasure references repeat births/pregnancies among adolescents onlybMeasure references family planning counseling or postpartum birth control planning as addressed during routine prenatal carecMeasure references multivitamin use in month prior to conceptionTable 2Sample of selected state Performance Measures and Priority Needs related to preconception healthSelected performance measures and priority needsPreconception health and health care Performance measure  •Percentage of women of childbearing age who receive preconception health care in the local health department Priority need  •Enhance preconception care and work toward eliminating disparities in infant and maternal morbidity and mortality  •Improve women’s preconception and interconception health  •Improve indicators of health in the preconception and perinatal periods, including but not limited to smoking, alcohol, folic acid use,   family violence, intention of pregnancy, access to and use of health care  •Promote sexual responsibility and reproductive healthNeural tube defects/folic acid Performance Measure  •Percent of women of reproductive age who consume at least 400 mcg of folic acid daily  •Percent of women of childbearing age taking folic acid regularlyBirth spacing and parity Performance Measure  •Percentage of live births to women who have another birth in less than 18 months  •Percentage of repeat pregnancies among adolescents 15–19 years of age Priority need  •Reduce repeat teenage pregnancies, unintended pregnancies and inadequate spacing of births through widely available, adequately    funded comprehensive, efficiently run family planning and support services  •Increase the proportion of births that are intended, including promotion of healthy interpregnancy spacingFamily planning Performance measure  •Proportion of low-income women who receive reproductive health/family planning services Priority need  •To improve access to and utilization of contraceptive servicesUnintended pregnancy Performance measure  •Percentage of pregnancies (live births, fetal deaths, abortions) that are unintended  •Percentage of women responding to the Pregnancy Risk Assessment Monitoring System (PRAMS) survey that they wanted to be   pregnant later or not then or at any time in the future Priority need  •Improve the health of children and families by increasing the percent of births that are intended  •Promote planned pregnancies and child spacingHealthy weight/obesity Performance measure  •Percent of women (18–44) with healthy weight (BMI)  •To increase the percentage of Family Planning clients with BMI greater than the 85th percentile who receive educational materials in    the Family Planning Clinics, and are referred to community sources Priority need  •To reduce proportion of women of childbearing ages, pregnant women and children and youth with BMIs in the underweight,    overweight and obese categories
Abstracts from the National Summit on Preconception Care
A total of 59 abstracts accepted for presentation at the National Summit on Preconception Care were reviewed and categorized according to primary area of focus. Approximately 32% addressed preconception health research; 27% described preconception care programs and activities; 22% outlined tools for provider or patient education; 15% detailed clinical practice strategies; and 3% highlighted policy-based strategies for increasing access to preconception care services.
Among the research abstracts, the topics noted most frequently pertained to methods for preconception risk assessment, potential predictors of adverse pregnancy outcomes, and variations in preconception health indicators across high-risk populations. Three abstracts described the use of the Perinatal Periods of Risk (PPOR) model [2] for assessing risks among various populations. Other issues included folic acid awareness among women and health care providers, maternal nutrition, pregnancy planning and risk behaviors, preconception hepatitis B prevention, and workplace hazards.
The PPOR model facilitates the identification of four contributors to infant mortality: maternal health and prematurity, maternal care, newborn care, and infant health. Excess rates of fetal and infant mortality in any of the four components indicate a need for targeted interventions within that construct [2–4]. CityMatCH’s 2000–2002 PPOR Practice Collaborative provided public health workers in 14 U.S. cities with the skills, knowledge, and support to implement the PPOR method in various urban communities [2]. The PPOR approach is also is being used statewide by Florida and Ohio and in selected areas across the country [2–4].
The Fountain Project in Kansas City, Missouri linked PPOR techniques with the Fetal Infant Mortality Review (FIMR) process in an effort to identify the causes of consistently higher rates of infant mortality among African-American residents compared with their non-Hispanic White counterparts. The findings of the analysis indicated an excess rate of maternal health or prematurity-related deaths among African-American infants. In light of the contributing social factors identified by the FIMR, the development of a Women’s and Children’s Wellness Center was proposed to provide social and clinical services for high-risk families (Cook BE, Guillory VJ, Cai J, Hoff GL, Manning J, unpublished data, 2005).
In an effort to facilitate the analysis of various perinatal health outcomes, a group of nine counties surrounding the San Francisco Bay (California) formed the Maternal, Child and Adolescent Health Bay Area Data Collaborative (BADC). The group used the PPOR model to evaluate recent county and regional fetal-infant mortality rates and reported substantial excess in the proportion of deaths among African-American infants, particularly in the prematurity and maternal health component of the model. Using a national comparison group, these investigators were able to determine that approximately 56% of the excess deaths among African-American infants could be prevented by targeting preconception and interconception risk factors (Stein EJ, Abramowitz A, Brown J, Chabra A, unpublished data, 2005).
Federally funded Healthy Start projects were highly represented among the 16 abstracts detailing preconception care programs and activities presented at the Summit. Of these, seven abstracts described the modification or expansion of existing programs to include direct services or community-based interventions for high-risk postpartum mothers or all women of reproductive age. The Magnolia Project in Jacksonville, Florida is an example of a Healthy Start program in which a comprehensive array of health services was offered in an effort to improve birth outcomes. Services included, but were not limited to, case management, education and risk reduction, and well-woman care. The target population was high-risk African-American women aged 15–44 years who lived in five zip code areas of Jacksonville. The project operates as a collaborative between the Northeast Florida Healthy Start Coalition, the Duval County Health Department, and local community-based organizations. As reported, a 2004 assessment of project services conducted by the Health Resources and Services Administration (HRSA) Office of Performance Review found high rates of success (>70%) in the resolution of key risks (for example, lack of family planning, and rates of repeat sexually transmitted diseases) among the project participants (Brady, CM, unpublished data, 2005).
The Missouri Bootheel Healthy Start project, a provider of referral and education services in a five county area of southeast Missouri, uses community-based education and interventions to improve adverse perinatal outcomes. The project has been one of few in the area that has promoted a family-focused approach to health education through the inclusion of various services directed at men. The curriculum for fathers has addressed a variety of issues, such as communication with the mother, addressing stress in the relationship, and the impact of nutrition on birth outcomes (Dean CG, Campbell T, Frazier V, Washington J, unpublished data, 2005).
Title X family planning clinics were identified as providers of preconception primary care and folic acid awareness interventions in two programs and activities abstracts. The Women Enjoying Life Longer (WELL) Project was initiated by the Maryland Department of Health and Mental Hygiene following a community needs assessment. Comprehensive preventive women’s health services were added to three Title X family planning clinics in Baltimore County, using Title X and Maternal and Child Health funding. Augmented services included nutrition and physical activity counseling, adult immunizations, smoking cessation interventions, and preconception counseling, as well as referrals for problems such as substance abuse, depression, domestic violence, and chronic disease. Early evaluation data indicated that patients and staff have responded positively to new services, patient knowledge of women’s health has improved, and patient volume has increased 37% (Cheng D, unpublished data, 2005).
The Oklahoma Birth Defects Registry developed, implemented, and evaluated a preconception “women’s health appraisal” project. The intervention was comprised of a 3-page health appraisal for women, with follow-up risk counseling, in selected family planning clinics (two rural and one urban). Pre-evaluation results indicated that 84% of nurses found the questionnaire helpful in assessing risk factors, 90% of nurses found it helpful as a guide to counseling and referrals, and 86% of patients increased their understanding of risk factors. Post-evaluation results found that 62% of patients modified one to three risk factors in a three-month period (Feuerborn VR, Pearson K, unpublished data, 2005).
The remaining programs were funded either through grants, private organizations, or state/local health departments and comprised comprehensive health care programs for women with a history of preterm delivery or a low birth weight infant and broad women’s wellness interventions aimed at improving women’s health or folic acid utilization. For example, the Interpregnancy Care Program at Grady Memorial Hospital in Atlanta, Georgia employs a “resource mother” to coordinate the provision of primary health care and dental services, enhanced nurse case management, and other outreach services to African-American women who delivered a very low birth weight infant at the hospital and who qualified for indigent or charity care. For 24 months postpartum, the women were offered health care visits every 1–3 months in order to address risks known to be associated with the delivery of a low birth weight infant such as poorly controlled chronic disease, short intervals between pregnancies, reproductive tract infections, periodontal disease, nutritional deficits, substance abuse, and stress. Preliminary evaluation of the 22 women retained in the pilot program indicated that approximately one-quarter of them were affected by unrecognized or poorly managed chronic health problems and none of the participants wanted to become pregnant during the next 2 years (Dunlop A, unpublished data, 2005).
Thirteen abstracts (22%) described the creation, dissemination, and evaluation of tools for patient or provider education. Of those, eight described marketing campaigns and toolkits for patient education, three specifically referenced provider education, and three assessed the use of women’s health appraisals by health care providers. For example, the California Preconception Care Initiative, Every Woman, Every Time, was created in 1989 through a partnership between Sutter Medical Center in Sacramento and the March of Dimes. The project conducted a metanalysis of the preconception care literature [5] and used this information in a consensus development process to produce a marketing packet for providers. Key components of the packet included the rationale for providing preconception care, a description of the essential elements of care, patient education materials, and information on billing methods. More than 9,000 packets were distributed statewide. An evaluation found that, among 187 providers responding, 75% indicated the information was very useful, 80% said they would distribute materials to patients, and 72% said they would use the billing codes provided. A further impact evaluation is being considered (Cullum AS, unpublished data, 2005).
A research project conducted at a New York inner-city hospital served by Albert Einstein College of Medicine/Montefiore Medical Center sought to evaluate the knowledge and awareness of providers regarding preconception care. A pre-intervention chart review and a provider survey were conducted to evaluate delivery or preconception care. The two-part intervention included: [1] a 1-h lecture for all providers, and [2] a standardized preconception care form inserted into all charts. A post-intervention chart review of a convenience sample and repeated provider survey were then conducted. The result was a significant improvement in documentation of the delivery of preconception care (p<0.05); however, provider knowledge and attitudes, as measured by the survey, did not change significantly (Bernstein P, unpublished data, 2005).
Clinical practice strategies were discussed in nine abstracts and included topics such as genomics, group care, screening for environmental exposures and maternal depression, smoking cessation, and provider knowledge of current practice guidelines. An example of a promising practice strategy presented at the Summit is the use of a group model of prenatal care to address topics relevant to preconception care. In 2002, the Comprehensive Family Care Center at Montefiore Medical Center implemented the group model of prenatal care developed by the Centering Pregnancy and Parenting Association. Traditional prenatal visits were replaced with group appointments lasting approximately 2 hours and attended by 10 to12 women. Typical clinical care services were provided during the visits and were supplemented with group discussions on pregnancy-related topics. Issues related to preconception health were covered during many of the meetings and included nutrition, substance abuse, contraception, and family planning. Since its inception, the group care model has been employed for 14 patient groups with high levels of patient and provider satisfaction reported, particularly related to enhanced opportunities for patient education (Bernstein P, Rising SS, Dolan S, Pardanani S, Merkatz IR, unpublished data, 2005).
Finally, policy-based strategies for the funding of preconception care were described in a small number of abstracts. One pair of abstracts highlighted Illinois’ efforts to promote preconception health using public policy and funding through Medicaid waivers, Title V, and other resources (Murphy AM, unpublished data, 2005; Saunders SE, unpublished data, 2005). Identified as a priority area, preconception care has been integrated into a number of programs, most notably the Illinois Healthy Women program. This Medicaid waiver program initiative extends coverage for family planning services to women who would otherwise lose their benefits after 60 days postpartum and to all women 19 through 44 years of age who were previously enrolled in Medicaid but who had lost their benefits (Murphy AM, unpublished data, 2005). Additional efforts have been made through the Illinois Family Planning Program. Preconception education is currently provided at all state-funded family planning clinics, with 16 state family planning agencies offering additional counseling and referral for high-risk clients [6]. Other strategies employed by the Illinois Department of Human Services include statewide genetic counseling programs and folic acid campaigns, screening mothers of children enrolled in the state’s Medicaid program for perinatal depression, and programs aimed at increasing birth spacing and promoting the health of teen parents (Saunders SE, unpublished data, 2005).
Conclusions
In public health, effective programs are those developed in response to a well-defined problem analysis, are mindful of the local context, and apply proven interventions. Although the scientific evidence in support of some preconception interventions is relatively well described within the literature, topics discussed during the National Summit on Preconception Care reframed the issues in light of new findings regarding unmet needs, health disparities, service delivery models, and tools such as the PPOR to assess risks among populations. Variations in the type and scope of policies, programs, and strategies were evident both in the diversity of the summit abstracts and in the state Title V indicators. Clearly, a preconception focus is being woven into existing programs and guiding the development of new initiatives. Innovative strategies are being implemented that target a variety of populations including community groups, health care providers, medical students, adolescents, and fathers. Finally, many state indicators are adopting a broader language to encompass the health needs of women of childbearing age and support the notion of a lifespan approach to preconception health.
It should be noted that the findings reported in this brief are limited by a number of factors. First, state measures and priorities reported by the federal Maternal and Child Health Bureau might not reflect the programs and projects actually being implemented by states and communities; they are only an expression of interest and concern. Next, the content of the abstracts accepted for the National Summit on Preconception Care presented a selection of current research, policies, and programs and, therefore, does not fully represent the scope of national preconception care projects. Finally, the identification of preconception health state Title V indicators required a certain amount of subjective interpretation regarding the actual focus of the Priority Need or Performance Measure. Although the authors have considerable knowledge of the definition and content of preconception care, it is possible that certain indicators might have been incorrectly excluded or included due to misinterpretation.
Notwithstanding these limitations, the results of the present analysis indicate a widespread recognition of the need for a continuum of care that starts well before birth, involves both men and women, and employs a lifespan approach to emotional and physical well-being. Because preconception interventions can take place at any time during a woman’s reproductive life and across a variety of dimensions, countless opportunities exist for positively influencing the health of women, children, and families. The variations among domestic preconception health projects and related public health priorities are reflective of the true breadth of the topic. If a national agenda for preconception health is to be moved forward, it is imperative that these efforts continue to be fostered and expanded in all settings and across a multitude of disciplines.

preconception care
reproductive health
pregnancy