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Universidade de São Paulo Faculdade de Saúde Pública Estado nutricional de vitamina A na gravidez e associação com desfechos materno-infantis no estudo MINA-Brasil

Neves PAR. Vitamin A status during pregnancy and its association with mother-infant outcomes in the MINA-Brazil study [thesis]. São Paulo: Faculdade de Saúde Pública da Universidade de São Paulo; 2018. Portuguese. Introduction Adverse exposures related to antenatal nutrition care have been associated with poor maternal and child outcomes. In this sense, vitamin A (VA) deficiency is a relevant public health issue for this vulnerable population, especially in developing countries. Objectives a) to investigate the predictors of serum retinol at the beginning of the third trimester of pregnancy (Article 1), b) to investigate associations between the VA status during pregnancy with the occurrence of maternal anemia and birth weight (Article 2), and c) to investigate the prevalence, as well as the associated factors with gestational night blindness and maternal anemia (Article 3) in Cruzeiro do Sul, Acre State, Western Brazilian Amazon. Methods Prospective birth cohort study (MINA-Brazil: Maternal and Child Health and Nutrition in Acre State), starting with recruitment of pregnant women. Between February/2015 and May/2016, pregnant women registered in antenatal clinics in the urban area of Cruzeiro do Sul were screened (n = 587) and two follow-up surveys were performed: the 1st one between 16 to 20 weeks of pregnancy and the 2nd one between 27 to 30 weeks of pregnancy. Socioeconomic, environmental, health history and lifestyle, anthropometric measures, blood samples, and ultrasounds data were gathered by the research team. Subsequently, between July/2015 and June/2016, daily visits to the municipal maternity hospital were performed in order to register all births and the outcomes of interest for this study. Multiple Poisson and linear regression models were used for statistical analysis, at P<0.05. Results In the Article 1 (n = 422), having a smoker in the household was inversely associated with serum retinol in pregnancy (β: -0.087; 95% CI: -0.166, 0.009); on the other hand, the seasonality (Amazonian winter β: 0.134; 95% CI: 0.063, 0.206), the weekly consumption of Amazonian carotenoid-rich fruits (β: 0.087; 95% CI: 0.012, 0.162), and serum retinol in the first assessment (β: 0.045; 95% CI: 0.016, 0.074) were positively associated with serum retinol at the beginning of the third trimester of pregnancy. In the Article 2 (n = 488), regardless the period assessed, VA deficiency was associated with the risk for maternal anemia (PR: 1.39; 95% CI: 1.05, 1.84), as well as it was inversely associated with maternal serum hemoglobin (β: -3.34; 95% CI: -6.48, 0.20), after adjustment for covariates. Similarly, inverse association was observed for the birth weight (β: -0.10; 95% CI: -0.20, -0.00), even though it was no longer associated after adjustment for plasma ferritin. In the Article 3 (n = 1,525), high prevalence rate of gestational night blindness (11.5%) and maternal anemia (39.4%) were observed. Associated factors with gestational night blindness were the number of people in the household (five or morePR: 2.06; 95% CI: 1.24, 3.41), smoking during pregnancy (PR: 1.78; 95% CI: 1.15, 2.78), and the completeness of less than six antenatal care visits (PR: 1.61; 95% CI: 1.08, 2.40). Associated factors with maternal anemia were being teenage (< 19 years PR: 1.18; 95% CI: 1.01, 1.38), gestational malaria (PR: 1.22; 95% CI: 1.01, 1.49), did not use any supplementation during pregnancy (PR: 1.27; 95% CI: 1.01, 1.62), and the number of antenatal care visits (< 6 visits PR: 1.40; 95% CI: 1.15, 1.70). Conclusion In a city in the Western Brazilian Amazon, the VA deficiency was associated with the risk for maternal anemia and was inversely associated with maternal serum hemoglobin and the birth weight. The current strategies and interventions targeting antenatal nutrition care must be reviewed in order to reduce the adverse effects for mother-child binomial caused by VA deficiency. Descriptors: Vitamin A Deficiency; Anemia; Birth Weight; Pregnancy; Longitudinal Studies.


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Varied food-sources provide VA to the human-beings, such as viscera, dairy products, eggs 41 (preformed VA esters, such as retinoid), and orange-yellow and dark-green vegetables (VA 42 precursors, mainly β-carotene) (7) . The bioavailability of VA precursors in the organism is less-43 efficient than for preformed VA esters, affecting thus the VA functions, depending on the food-44 matrices which the vitamin was obtained (7,8) . Hepatic storage is the main site where VA can be 45 found in the humans, and varied methods were developed attempting to measure retinol stores in the 46 organ, being the serum retinol (µmol/L) the widest used and recommended method in 47 epidemiological surveys (7) .

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VA deficiency is still an important public health problem in low-and middle-income 49 countries. The global prevalence of VA deficiency during pregnancy is estimated to be 15.3%, 50 considering serum retinol concentrations < 0.7 µmol/L, and 7.8% based on reported frequency of 51 night blindness (7) . The latest global estimates for pregnant women in Latin America and the 52 Caribbean showed a prevalence of 2% of low serum retinol and 4.4% of night blindness, however 53 some estimates were obtained through regression-based models due to the lack of available data for 54 most countries in the region (7) . In Brazil, a national representative study with childbearing-age 55 women (15-49 years) found a prevalence of 12.3% of VA deficiency, and an alarming prevalence 56 of VA insufficiency (serum retinol < 1.05 µmol/L) of 49.2% (9) . Thus, the identification of the 57 predictors of VA status during pregnancy may help in the prevention of inadequate VA nutritional 58 status, targeting safe interventions for improvement of VA status during this period. Considering 59 the supplementation during pregnancy might be harmful to the mother-child pair owing to 60 teratogenic effects if higher than 10,000 IU daily or 25,000 IU weekly (10) , other strategies to prevent 61 VA insufficiency or deficiency must be considered.

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Poor VA status in pregnancy is caused mainly by inadequacies on dietary intake of VA-rich 63 foods, as well as during infectious episodes (7) . Alternatively, zinc and iron deficiencies have been 64 reported to occur concomitantly with VA deficiency, due to metabolic pathways shared by these 65 micronutrients (11,12) . A national analysis in Brazil compared nutrient intake among pregnant women, through two non-consecutive food records, showing elevated inadequacies for iron (97%), VA 67 (71%), and zinc (35%), according to Estimated Average Requirements (13) .

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There is a lack of studies on the predictors of VA status at the beginning of the third trimester 69 of pregnancy, a crucial moment when the maternal VA status has a positive correlation to the 70 baby's liver stores in the first six months after birth (14) . Likewise, there is no prospective study in Census, only 12.7% of the households had access to proper sanitation in Cruzeiro do Sul (15) . were invited to participate in our study through phone calls or home visits. Afterwards, a home-visit 95 was scheduled to obtain written consent and to perform the socioeconomic and health interview.

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Twin pregnancies were excluded from this analysis. set the GA precisely, which was then compared with GA based on LMP (16) . The best GA estimate 120 was obtained based on the following criteria: the LMP was used when participants reported regular 121 menstrual cycles, no use of hormonal contraceptive methods, and when agreement between LMP 122 and ultrasound estimates of GA was ≤7 days for pregnancies <20 weeks or ≤14 days for 123 pregnancies from 20-28 weeks; ultrasound measurements were used in all other cases.  of blood collection by a portable haemoglobinometer (Hemocue ® Hb301; Angelholm, Sweden) (17) .

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The serum samples were collected in a dry test tube, protected from light and centrifuged within 2 Packard, Palo Alto, California, USA) (18) . At the second evaluation, C-reactive protein (CRP) was 139 measured as a marker of acute inflammation, using an IMMAGE Immunochemistry System 140 (Beckman Coulter, Brea, CA, USA). Biochemical data were double-typed using Excel for Windows 141 and then converted to Stata 14.0 for data processing and analysis. Intra-and inter-assay CVs were < 142 7%.

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Anthropometric assessment 145 Weight and height were measured by trained researchers according to standardized procedures (19,20) . guidelines from the Institute of Medicine based on the pre-pregnancy BMI (21) . Amazonian scholars was adapted for this study (22) . The frequency of food groups consumption 165 (dairy, beans, green and root vegetables, fruits, Amazonian fruits, eggs, meat, viscera, fish, cereals 166 and breads, oils, and ultra-processed) was estimated during the last month, with the following 167 response options: never, 1-3 times/month, 1-3 times/week, 4-6 times/week, 1 time/d, 2-3 times/d, ≥ 168 4 times/d (22) . For this analysis, we considered some VA rich food, grouped as follows: fruits and 169 vegetables (no/monthly, weekly, or daily consumption), Amazonian fruits (e.g., Açaí -Euterpe  Mey -no/rare or weekly consumption), viscera (e.g., liver, gizzard, heart -no/rare, monthly or 175 weekly consumption), fish (no/monthly, weekly, or daily consumption), dairy (no/monthly, weekly, 176 daily, or ≥ 1 times/d consumption), and eggs (no/monthly, weekly, or daily consumption).   Table 2 shows the comparisons of study participants between the two evaluations. consumed Amazonian fruits weekly than among those who consumed these fruits less often.

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Conversely, having a smoker in the house presented a negative correlation, with mean serum retinol 245 of 0.087 µmol/L lower when compared to pregnant women without a smoker in the house. These 246 four variables were responsible for explaining 11.1% of the variability in serum retinol (Table 3).

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Control for CRP concentrations had a non-significant effect on the R 2 , lowering it. Hence, we did

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Literature on determinants of serum retinol in mid-gestation is scarce; most available data 262 analysed either the determinants of retinol in the final gestational weeks, or VA deficiency during 263 pregnancy or postpartum (24,25) . This study is the first to address this subject in this specific period in 264 a less-developed area of Brazil, which is relevant in view of the physiological characteristics of the 265 forthcoming gestational weeks. Throughout the third trimester, foetus growth occurs rapidly and 266 thus seems to increase the need for specific nutrients to meet foetal needs, such as VA (14) . 267 Furthermore, it is in this trimester that the mother-to-child transfer of nutrients may occur more 268 intensely, highlighting the necessity of achieving good foetal stores of VA in the liver in this period 269 (14,24) . Thus, having a good nutritional status prior to the third trimester might prevent inadequate 270 mother-to-child transfer of VA and/or insufficient storage of retinol in the foetal liver, which is 271 essential to complement the VA supply provided by human milk in the first six months of life (14) . 272 Moreover, pre-pregnancy nutritional status has a crucial role in maintain optimal concentrations of 273 micronutrients throughout pregnancy, evidencing that optimal women's nutritional status has to be 274 reached before the establishment of pregnancy (26) .

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In this study, average of serum retinol at the second evaluation was higher than at the first 276 evaluation, being more than 75% of our sample presented retinol concentrations above 1.05 µmol/L 277 at the second point measured in pregnancy, which is a cut-point to insufficiency in VA (7,14) . It is 278 important to note that plasma volume during pregnancy increases until the last trimester, 279 consequently declining the concentration of nutrients, even though it is less than the major changes 280 in plasma volume (27) . Therefore, the total amount of micronutrients in circulation increases during 281 pregnancy (27) .

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Additionally, the importance of VA liver stores to maintain the homeostasis of serum retinol 283 must be recognized. Under adequate VA nurture conditions, the liver is the main site of retinol 284 storage in the body, and this regulates VA homeostasis, keeping adequate concentrations of retinol 285 in the blood stream. Furthermore, the VA status indirectly regulates bioconversion of carotenoids to 286 retinol, as well as the diet-responsive regulatory network (27) . Thus, even when the intake of VA does 287 not attend the recommendations, the serum retinol is influenced by the homeostasis, bioconversion 288 of carotenoids or even the inflammatory status (12) . This fact may be one of the reasons our final 289 multiple regression models have explained only 11.1% of the variability in serum retinol. It is 290 possible that majority of the women in our study had adequate pre-pregnancy nutritional status of 291 VA, considering the mean values of retinol in both evaluations were higher than in other 292 studies (24,25,28) . were associated with poverty and race (Hispanic and black) (28) . In a large cross-sectional study of 298 women within all gestational trimesters in Guinea-Bissau, the mean serum retinol was 1.03 (SD: 299 0.33) µmol/L, and the GA ˃ 20 weeks was consistently associated with ~0.1 µmol/L lower serum 300 retinol (β: −0.09 to −0.11) compared with GA from 7 to 16 weeks (24) . Another cross-sectional study 301 of post-partum women in an impoverished area of South Africa, where pregnant women frequently 302 consume liver in their diet, found a mean serum retinol of 1.03 (SD: 0.4) µmol/L (25) . These studies 303 raise a few questions, such as the influence of pre-pregnancy nutritional status of VA, 304 socioeconomic level, food source availability, and supplement use, among others. Also, our results 305 reinforce the role of homeostasis acting on serum retinol even in low-income areas, like the 306 Brazilian Amazon.

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Even considering the absorption rate and bioconversion factor to retinol, carotenoids are an 308 important source of VA, especially for populations from low-and middle-income countries (29) . In 309 Western Kenya, enhanced nutrition education and intake of orange-fleshed sweet potato by 310 pregnant women through nine months postpartum was positively associated with higher intakes of 311 VA, and reduced odds of low serum retinol binding protein, compared with participants who 312 received standard-of-care approaches (30) . Amazonian fruits such as buriti and pupunha are 313 extremely rich in carotenoids, either through the consumption of the fruit or their oil. Each 100 g of 314 buriti has 4,104 mg of carotenoids, while the same amount of pupunha has 1,500 mg (31) . It is 315 relevant to point out the availability of carotenoids, and how these Amazonian fruits are usually 316 consumed. Carotenoids from fruits are more available than vegetables owing to the weak layer in 317 the fruit cells. Additionally, heating up helps release carotenoids from their matrix (8) . These two 318 Amazonian fruits are normally cooked before consumption, which may foster improved nutrient 319 availability. Experimental studies with rats have shown the improvement on serum and liver VA 320 content with the consumption of buriti oil, which might be extended to humans (32,33) .

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The availability of foods and crops relates to intake and nutritional status of many nutrients, 322 including VA. In our study, seasonality was one of the main factors positively associated with 323 serum retinol. Seasonal patterns of vegetable and fruit production can potentially affect dietary VA 324 intake (34) . This finding is in line with those of other studies, which showed that the time of year may 325 affect the intake of nutrients, possibly due to the availability and affordability of food in some 326 seasons (32,33) . In Manila, Philippines, a prospective study with young women showed that in specific 327 periods the consumption of fruits is higher than in others (35) . Another prospective study in South 328 Africa showed that the consumption by pre-school age children of pumpkin, butternut squash, and 329 orange-fleshed sweet potato was higher during rainy months (34) . The Amazon rainy season is the 330 period of the year when Amazonian carotenoid-rich fruits are most available, enhancing the 331 nutritional status of VA in our population (31) .  (36,37) . Taking into account that most of our sample had serum retinol above 1.05 µmol/L, 340 we considered the retinol stores were replete for the majority of participants. In addition, with the 341 contribution of Amazonian fruits, the nutritional status of VA for most of our population was 342 adequate, elucidating how important the nutritional status of VA is in the first half of pregnancy to 343 foster good retinol levels in the final gestation period. This could offer a good supply of VA to the 344 foetus prior to the last trimester, an important period of mother-to-child transfer of VA, as well as 345 preclude clinical manifestation of VA deficiency (xerophthalmia) and its consequences, which are 346 more often seen in the last trimester of pregnancy in low-income areas (38) .

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Our results show that having a smoker in the house impacts negatively on serum retinol.

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In this study, other covariates were not associated with the VA status in the final multiple 356 model. In a cohort study in England, serum retinol in pregnancy was negatively associated with 357 bone mineralization in new-borns (5) . Thus, considering the well stablished role of vitamin D in the 358 bone health (42) , we hypothesized that the VA and vitamin D might share metabolic pathways still 359 unknown, which might influence on adequate nutritional status of both vitamins. However, we did 360 not observe any association between these vitamins. Inadequate VA status in pregnancy is more 361 likely to occur in less-advantaged populations, evidenced by studies in the United States (28) and 362 Brazil (43) . Additionally, some episodes during the reproductive history of the pregnant women might 363 affect their VA status, such as history of abortion, lowering the liver retinol stores for subsequent 364 pregnancies (43) . Despite it all, no association with VA status at mid pregnancy was verified for some 365 covariates related to socioeconomic and reproductive aspects above mentioned. Furthermore, 366 previous episodes of malaria can affect subject's VA status, as during the reproductive cycle and 367 metabolism of the Plamosdium malaria the host's liver is affected. In this sense, frequent episodes 368 of malaria through the life course may impair adequate retinol stores, as well as raise concentrations 369 of acute phase proteins, like CRP (44) . Nevertheless, neither the history of malaria nor the CRP 370 concentrations were associated with the VA status in pregnancy in this study.

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Limitations of the study include the lack of data on other biochemical indicators of VA 372 status, e.g. retinol binding protein; the food frequency questionnaire did not include amounts of 373 food intake, precluding estimates of nutrient intake by the participants; and the fact that our study 374 sample was not representative of all pregnant women living in both rural and urban areas, with 375 losses to follow-up were higher than we expected, mainly because of difficulties in contacting 376 participants to attend evaluations. As for strengths, the prospective study design allowed us to 377 assess causality, the measurement of serum retinol used the HPLC assay, recommended by World        Results: A total of 488 mother-newborn pairs were surveyed. Combined VA deficiency 12 status increased the risk for maternal anemia (adjusted prevalence ratio: 1.39; 95% CI:    cell differentiation occurs rapidly, is essential for fetal growth and maternal metabolism 36 [1][2][3]. Retinol may act in early embryo development, differentiation, and organ 37 maturation of a countless number of cells and tissues as pregnancy progress [4,5]; β-38 carotene has an antioxidant role in humans, being associated with the reduction of 39 oxidative stress and hence with the lower risk of restricted fetal growth [6]. 40 Nonetheless, vitamin A deficiency (VAD) is still one of the most prevalent nutritional 41 deficiencies worldwide, affecting mostly pregnant and lactating women, and pre-school 42 children [3]. According to the latest WHO estimates for the period of 1995-2005, the 43 prevalence of VAD in pregnancy in low-and middle-income countries (LMIC) was 44 15.3%, with more than 19 million women affected [3].

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However, the quality of the evidence was considered moderate and the heterogeneity 53 was significant among the studies [10][11][12].

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The evidence for possible associations between maternal VA status and newborn 55 BW is still unclear. In high-income countries (e.g., Canada and United Kingdom), 56 where gestational serum retinol was found to be higher when compared with LMIC, 57 serum retinol concentrations during pregnancy were inversely associated with BW, 58 whereas a positive effect of serum β-carotene was found [6,9,13]. In Southern Ethiopia, 59 a cohort study found that VAD was not associated with low BW (LBW) (RR: 1.27; 60 95% CI: 0.86, 1.87) [14]. Two meta-analysis of randomized trials in LMIC found 61 contradictory effects for oral gestational VA supplementation on LBW [10,11].

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Anemia is the most prevalent nutritional deficiency worldwide, affecting about 63 41.8% of all pregnancies [8]. Observational and meta-analysis studies to date have 64 shown benefits of higher serum VA concentration during pregnancy on maternal 65 anemia, and unclear effects for newborn BW. Thus, as maternal VA status remains a 66 relevant public health concern in developing countries, further assessments of possible 67 effects of VA status during pregnancy are important for defining future strategies for 68 antenatal care and advancing the current scientific knowledge [15].

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In the present study, we investigated the relationship between gestational 70 biomarkers of VA status (serum retinol and β-carotene) with maternal anemia and 71 newborn BW in a prospective cohort study in the Western Brazilian Amazon. We 72 hypothesized that optimal VA status during pregnancy could modify the risk for 12.7% of the households had access to proper sanitation in Cruzeiro do Sul [16,17]. 86 Further, the municipality is located in the main malaria endemic hotspot in Brazil, the 87 Juruá River Valley [18,19]. May 2016, at about 28 weeks of pregnancy, based on the best estimate of GA, as 108 described elsewhere [18].

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A wealth index was constructed as a proxy of the socioeconomic status of the 126 participant's family based on the ownership of assets by the household. We applied 127 principal component analysis [20] and used the first component (which explained 128 18.9% of the variation between the households) to generate the index. The quintile 129 distribution of the index was included in the analysis.

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During both assessments we registered maternal anthropometry (pre-pregnancy 131 weight and height), smoking during pregnancy, biochemical measurements (serum 132 retinol, µmol/L; β-carotene, µmol/L; vitamin D, 25-hydroxivitamin D3, ng/mL; ferritin, 133 µg/L; C-reactive protein -CRP, mg/L; and hemoglobin concentrations -Hb, g/L), and 134 conducted the ultrasound exam. Maternal height was measured to the nearest 0. 1 cm, 135 and the pre-pregnancy BMI (kg/m 2 ) was calculated following WHO recommendations 136 [21,22]. As the reporting of smoking was very low in each assessment (< 4%), we 137 considered a dummy variable to indicate smoking either in one or both assessments. concentrations of retinol, β-carotene, and vitamin D were measured by using HPLC 165 methods (HP-1100 HPLC system, Hewlett Packard, Palo Alto, California, USA) [24]. 166 Gestational Hb was determined at the time of blood collection by a portable 167 hemoglobinometer from Hemocue ® (Hb301; Angelholm, Sweden). Gestational anemia 168 was defined as Hb < 110.0 g/L in each assessment [8]; subsequently, one categorical 169 variable was created indicating no anemia in both assessments, anemia at least in one 170 assessment, or anemia in both assessments. Plasma ferritin concentrations were 171 measured by enzyme immunoassays (Ramco, Houston, TX) and the cut off adopted for 172 iron deficiency was < 15 µg/L [25]. CRP was measured by an IMMAGE 173 Immunochemistry System (Beckman Coulter, Brea, CA, USA), and concentrations ≥ 5 174 mg/L were adopted as acute inflammation [26]. Plasma ferritin and CRP concentrations 175 were only available for the second assessment. Intra-and inter-assay CVs were < 7%.

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At delivery, venous blood samples were collected, and maternal Hb was determined by 177 an automated cell counter (Labtest SDH-20, Lagoa Santa, Brazil).

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Outcomes and exposures of interest, and data analysis 180 Maternal outcomes were Hb and anemia (Hb < 110,0 g/L) at delivery [25], and 181 the newborn outcomes were the BW (kg) and the BW z-score, according to Intergrowth 182 21 st Project [27].

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As the main exposures, we considered the serum retinol and β-carotene  [8,27,28], two different models were tested: model 1 (control for newborn sex and 226 gestational age at delivery, and adjusted for covariates) and model 2 (further adjustment 227 for plasma ferritin concentrations). Control for GA at delivery was not performed to 228 BW z-score [27].

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The effect of each VA status marker on each outcome was considered significant 230 when P < 0.05. Missing data were included in the multiple models by creating missing-231 value categories. The fit of the model was ascertained by an examination of residuals, 232 which did not show any potential harmful effect. Collinearity was examined by the 233 correlation matrix. All analyses were done in Stata 14 (StataCorp,College Station,234 Texas, USA).  Tables 1 and 2. We observed that 27.4% of participants were teenagers, 248 and less than 70% had either proper access to water supply or to sanitation facilities 249 ( Table 1). The average pre-pregnancy BMI was 23.4 ± 4.3 kg/m 2 (n = 530); 60.3% of 250 participants used only iron-folic acid supplements, while 34.7% used multiple 251 micronutrients with VA (n = 527). Overall, 74.6% of women did not present with 252 anemia in any assessment, and 18.2% presented anemia in one of the two assessments 253 (n = 452). Frequencies of iron deficiency and acute inflammation were 42.4% and 0.2% 254 (n = 464), respectively (data not shown in Tables).

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In the crude analyses, VAD in the first assessment, as well as the combined 256 VAD and the combined VA insufficiency, were negatively associated with Hb at 257 delivery. VAD in the first assessment, combined VAD, and combined VA insufficiency 258 increased the risk for maternal anemia at delivery; VAD in the second assessment was 259 not associated with maternal anemia. Variations of serum retinol and β-carotene 260 concentrations measured as the difference between assessments or tertiles of the 261 differences were not associated with the maternal and newborn outcomes. Neither of 262 these exposures was associated with newborn BW outcomes (Online Resource 1).

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For maternal anemia at delivery, VAD in the first assessment, combined VAD, 264 and combined VA insufficiency were associated with the risk for anemia in model 1.

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Continuous VA exposures were not significantly associated with maternal Hb at 269 delivery. In contrast, combined VAD was negatively associated with maternal Hb at 270 delivery in both models. Combined VA insufficiency in model 1 was negatively 271 associated with maternal Hb at delivery, yet in model 2 this association was no longer 272 significant (Table 4).

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There was a negative association between combined VAD and newborn BW.

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After further adjustment for plasma ferritin (model 2), the association was smoothed, 275 and no longer significant (Table 5). There was no association in the adjusted models for 276 newborn BW z-score (Online Resource 2). In this prospective cohort study in the Western Brazilian Amazon, we showed 280 that VA status during pregnancy was associated with maternal anemia and Hb, as well 281 as newborn BW. The occurrence of VAD during pregnancy was associated with an 282 increased risk for maternal anemia at delivery of 39%. In a condition without gestational 283 anemia, VAD and/or VA insufficiency in different moments during pregnancy were 284 also associated with increased risk for maternal anemia at delivery. A deleterious effect 285 of combined VAD was also seen for maternal Hb at delivery. Finally, a negative effect 286 was seen for BW of babies born to women who presented combined VAD, yet after 287 further adjustment for iron deficiency status the effect was smoothed. It must be 288 highlighted that these results were observed after controlling for a set of confounders 289 [2,25].

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Our hypothesis was only confirmed for retinol concentrations in pregnancy, as 291 we did not observe any effect for β-carotene in this population. It is likely that we have 292 not seen any effect for serum β-carotene because it is a less active and effective source 293 of VA when compared with serum retinol [5].

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The mechanisms by which VA affects anemia and Hb concentrations remain 295 unclear, although some hypotheses can be outlined: 1) VA plays a role in iron 296 metabolism, mobilizing the mineral from their hepatic stores; 2) the role of VA on 297 erythropoiesis; 3) VA enhances iron absorption in the gut; and 4) VA decreases the 298 inflammatory status, hence also decreasing the risk of anemia [10,25,29,30]. Anemia is 299 a condition affecting mainly pregnant women and children worldwide, and it is 300 associated with poor birth outcomes (including LBW and preterm births), as well as 301 maternal mortality [25]. Roughly, about 50% of anemia cases are caused by iron 302 deficiency [25], and strategies to reduce its burden are recommended, for example iron-303 folic acid supplementation during antenatal care [8]. However, it seems important to 304 invest in alternatives to boost other strategies that are already in practice. In this sense, 305 our results, in consonance with meta-analyzes conducted in LMIC [10][11][12], strengthen 306 the evidence that decreasing VAD in pregnancy consequently improves maternal Hb as 307 well as reduces the prevalence of anemia and its associated harmful effects during 308 pregnancy and at delivery [25].  [10,11,14]. Thus, our results suggest that inadequacies in gestational VA status might 313 negatively affect BW, yet after adjustment for plasma ferritin the association smoothed.

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Our hypothesis is that VAD acts as a limiting factor of optimal in-utero growth through 315 pathways that need to be clarified. Conversely, we did not observe associations between 316 any marker of VA status and BW z-scores. Therefore, the relation between gestational 317 VA status and BW is still unclear.

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Prior studies in high-income countries have shown disagreement with our 319 findings in relation to BW. Handel et al. [13] in Southampton, UK, found that retinol 320 measured in late pregnancy (~34 weeks of gestation) negatively influenced BW, as each 321 unit increment of retinol was responsible for decreasing the BW by 110 g, adjusted for a 322 set of potential confounders. In Portsmouth, UK, Mathews et al. [9] obtained two 323 measurements of serum retinol throughout pregnancy, similarly to our study (first 324 assessment ~16 weeks and second assessment ~28 weeks of pregnancy), and found that 325 the serum retinol at the second moment decreased the BW (β: -208.4 g; p < 0.001), yet 326 they only adjusted the model for maternal height and smoking. They also explored the 327 variation of retinol concentrations during pregnancy, as we did, encountering that large 328 reductions of retinol, as the pregnancy progressed, were responsible for bigger infants 329 (P = 0.002), though, unfortunately, they did not show any coefficient regression in the 330 paper for this finding. In Montreal, Canada, a case-control study by Cohen at al. [6] 331 encountered that retinol measured between 24-26 weeks of pregnancy increased the 332 odds for small-for-gestational-age (adjusted OR: 1.41; 95% CI: 1.22, 1.63).

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In this study, although the prevalence of VAD in both assessments was not too 334 high when compared with other settings [31], comparable to values in high-income 335 countries [6,9,13], important discrepancies between our study setting and high-income 336 countries must be addressed. Northern Brazil still faces issues in some socioeconomic 337 and environmental characteristics, being a less-advantaged region in the country [32].

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Poor micronutrient status during pregnancy is an important risk factor for a 348 range of deleterious outcomes to the mother-baby binomial [15]. Therefore, considering 349 the social vulnerability of the population in Northern Brazil plus the negative effects of 350 VAD and anemia evidenced by our study, strategies addressing nutrition and health 351 assistance during pregnancy should be reviewed. There is some evidence of the benefits 352 of multiple micronutrients (MMN), containing VA and iron-folic acid, delivered in 353 antenatal care for some outcomes included in our study [28,36]. A recent Cochrane 354 meta-analysis pointed out that MMN supplementation during pregnancy reduced the 355 risk of LBW (RR: 0.88; 95% CI: 0.85, 0.91) [36]. Smith et al. [28] found that MMN 356 supplementation reduced the risk of LBW (RR: 0.81; 95% CI: 0.74, 0.89), and for 357 anemic women, reductions in small-for-gestational-age births were observed (RR: 0.92; 358 95% CI: 0.87, 0.97). In all studies, comparison was made with iron-folic acid 359 supplementation.

360
Our study has limitations. We had losses to follow-up, potentially due to logistic 361 constraints faced by the research team in attempting to contact the participants for 362 enrollment in the study and to schedule the assessments, which are marked features in 363 this area (i.e., poor internet connection, lack of street labels for many addresses, and 364 intermittent mobile signal). The possibility of unknown confounding factors cannot be 365 excluded as we work with observational data. Despite these limitations, the strengths 366 must be highlighted, including: 1) the prospective design of the study, with two 367 measurements of potential exposures throughout pregnancy; 2) the biochemical 368 assessments of the main exposures followed standardized laboratory procedures to        [25]; f Difference in biochemical indicators between assessments (2 nd assessment -1 st assessment); g Deficiency or insufficiency during pregnancy combined: in at least one or both assessments. .51) a Hemoglobin < 110,0 g/L; b 1 st assessment: between 16-20 weeks of pregnancy/ 2 nd assessment: ~28 weeks of pregnancy; c For 'deficient during pregnancy' deficiency (retinol < 0.7 µmol/L) in at least one assessment or both was considered / for 'insufficient during pregnancy' insufficiency (retinol < 1.05 µmol/L) in at least one assessment or both was considered; d Model 1: iron deficiency 2 nd assessment (plasma ferritin < 15 µg/L), gestational malaria (no or yes), gestational supplementation (no supplementation, acid folic + iron, multiple micronutrients with vitamin A), number of antenatal care appointments (< 6, 6-8, or ≥ 9), pre-pregnancy body mass index (underweight, normal weight, overweight, or obesity), gestational age at delivery (weeks), number of live births (none, 1-2, or ≥ 3), number of people living in the household (1-2, 3, 4, or ≥ 5), wealth index (quintiles), Bolsa Família cash transfer program receipt (no or yes), maternal age (< 20 or ≥ 20 years); e Model 2: Model 1 with further adjustment for gestational anemia (no anemia during pregnancy, anemia at least in one assessment, or anemia in both assessments); f Adjusted prevalence ratio; g P for trend; * P < 0.05; ** P < 0.01. Insufficient during pregnancy -3.28 (-5.73; -0.82) * -2.28 (-4.61; 0.04) a Hemoglobin in g/L; b 1 st assessment: between 16-20 weeks of pregnancy/ 2 nd assessment: ~28 weeks of pregnancy; c µmol/L; d Difference in biochemical indicators between assessments (2 nd assessment -1 st assessment); e For 'deficient during pregnancy' deficiency in at least one assessment or both was considered / for 'insufficient during pregnancy' insufficiency in at least one assessment or both was considered; f Model 1: plasma ferritin 2 nd assessment (µg/L), gestational malaria (no or yes), gestational supplementation (no supplementation, acid folic + iron, multiple micronutrients with vitamin A), number of antenatal care appointments, pre-pregnancy body mass index (kg/m 2 ), gestational age at delivery (weeks), number of live births, number of people living in the household, wealth index (quintiles), Bolsa Família cash transfer program receipt (no or yes), maternal age (years); g Model 2: Model 1 with further adjustment for gestational anemia (no anemia during pregnancy, anemia at least in one assessment, or anemia in both assessments); * P < 0.05; ** P < 0.01. Insufficient during pregnancy -0.00 (-0.08; 0.06) -0.01 (-0.09; 0.06) a Birth weight in kg; b µmol/L; c 1 st assessment: between 16-20 weeks of pregnancy/ 2 nd assessment: ~28 weeks of pregnancy; d Vitamin D in the 1 st assessment; e Vitamin D in the 2 nd assessment; f Difference in biochemical indicators between assessments (2 nd assessment -1 st assessment); g Difference in vitamin D concentrations in both assessments (2 nd assessment -concentrations in1 st assessment); h For 'deficient during pregnancy' deficiency in at least one assessment or both was considered / for 'insufficient during pregnancy' insufficiency in at least one assessment or both was considered; i Model 1: controlled for newborn sex and gestational age at delivery; adjusted for total gestational weight gain (insufficient, adequate, or excessive), type of delivery (vaginal or cesarean section), pre-pregnancy body mass index (kg/m 2 ), preterm birth (no or yes), gestational anemia (no anemia in both assessments, anemia in at least one assessment, or anemia in both assessments), smoking during pregnancy (no or yes), number of prenatal care appointments, gestational malaria (no or yes), vitamin D concentrations (ng/mL), number of live births, number of rooms in the household, maternal age (years), and wealth index; j Model 2: Model 1 further adjusted for plasma ferritin in the 2 nd assessment (µg/L); * P < 0.05.   [25]; b µmol/L; c 1 st assessment: between 16-20 wks of pregnancy/ 2 nd assessment: ~28 wks of pregnancy; d Vitamin D in the 1 st assessment; e Vitamin D in the 2 nd assessment; f Difference in biochemical indicators between assessments (2 nd assessment -1 st assessment); g Difference in vitamin D concentrations in both assessments (2 nd assessment -concentrations in1 st assessment); h For 'deficient during pregnancy' deficiency in at least one assessment or both was considered / for 'insufficient during pregnancy' insufficiency in at least one assessment or both was considered; i Model 1: controlled for newborn sex and gestational age at delivery; adjusted for total gestational weight gain (insufficient, adequate, or excessive), type of delivery (vaginal or cesarean section), pre-pregnancy body mass index (kg/m 2 ), preterm birth (no or yes), gestational anemia (no anemia in both assessments, anemia in at least one assessment, or anemia in both assessments), smoking during pregnancy (no or yes), number of prenatal care appointments, gestational malaria (no or yes), vitamin D concentrations (ng/mL), number of live births, number of rooms in the household, maternal age (years), and wealth index; j Model 2: Model 1 further adjusted for plasma ferritin in the 2 nd assessment (µg/L).

Introduction
Vitamin A deficiency (VAD) and anaemia are among the main nutrition-related problems of public health concern worldwide, especially in vulnerable populations in lowresource settings, such as women of childbearing age or preschool children [1][2][3] . Both VAD and anaemia have been considered important determinants of poor maternal and child outcomes, such as impaired growth and development in utero, premature delivery, low birth weight, infectious diseases, child development, and maternal and child mortality 2-6 .
Depending on its severity, VAD can progress to clinical ocular symptoms, leading to permanent visual impairment if not treated, named xerophthalmia. The first clinical manifestation of xerophthalmia that can be assessed is night blindness, a condition in which one cannot see in low-light conditions 7 .
The latest estimates from the World Health Organization (WHO) indicate that the global prevalence of gestational night blindness (GXN) in populations at risk of VAD is 7.8% (9.75 million pregnant women), and rates ≥ 5% are considered of public health concern. In Brazil, the public health significance of GXN was considered mild (3.5%); however, the estimates for Brazil were retrieved through regression-based methods, owing to a lack of available data in the country 3 . Also, regional data suggest that prevalence of GXN in the country might be underestimated, as a prevalence of 9.9% was observed in a maternity hospital in Rio de Janeiro city 8 .
According to WHO, 41.8% of all pregnancies worldwide are affected by anaemia 4 , and half of these cases are caused by iron deficiency 2 . The latest official Brazilian data show a prevalence of anaemia of 29.4% in women of childbearing age, reinforcing the hypothesis that a considerable number of women will start their pregnancies iron-deficient, increasing the risk of anaemia in pregnancy 9 .
Among the principal causes of VAD and anaemia in pregnancy is the inadequate dietary intake of foods rich in vitamin A (VA) and iron, along with high rates of infection, and aspects related to the physiology of pregnancy, such as the haemodilution in the third trimester 3 . Other causes of VAD and anaemia include poor socioeconomic and environmental conditions, maternal illiteracy, history of abortion, and inadequate antenatal care 3,8,10 .
Most evidence on GXN and maternal anaemia in Brazil does not come from population-based studies, and there is a lack of new studies in less-advantaged areas. Thus, we aimed to describe the prevalence and the factors associated with GXN and maternal anaemia in a population-based cross-sectional study in the Brazilian Amazon.

Population and study site
The present cross-sectional analysis refers to the baseline assessment of the MINA-Brazil Study (Maternal and Child Health in Acre, Brazil), which is a large, population-based prospective birth cohort study in the municipality of Cruzeiro do Sul, Acre State, Western Brazilian Amazon. Cruzeiro do Sul has few more than 80,000 inhabitants, mostly living in the urban area of the municipality (70%), and is the second largest city in Acre State, situated All patients living in the municipality were considered eligible to the study, independent of the place of residence (urban or rural). The research team visited mothers within the first 12 hours after delivery, before hospital discharge, to explain the study protocol and to invite for participation. Upon acceptance, an interview was held in order to collect data on socioeconomic, environmental, obstetric history, and gestational characteristics. Tablets with CSPro software (https://www.census.gov/programs-surveys/international-programs.html) were used for data-entry. The analysis was restricted to singleton deliveries.

Outcomes of interest and covariates
The first outcome of interest was GXN, assessed using the standardized interview proposed by WHO and adapted to the Brazilian context 16,17 . This interview is composed of three questions on the subject's visual capacity in daylight and dim light, taking the visual capacity prior pregnancy as reference, as follows: 1) do you have difficulty seeing during the day? 2) do you have difficulty seeing with decreased light or at night? 3) do you have night blindness? A case of GXN was ascertained when the participant responded NO to the first question and YES to either the second or third question, or both. Women with any visual impairment were asked about their visual ability using glasses or contact lens 17 . To facilitate the comprehension of questions, simple language and examples of places with dim light in the municipality were used 16 . The second outcome assessed was maternal anaemia, classified as maternal haemoglobin at delivery < 110.0 g/L at sea level 2 . Venous blood samples were collected and maternal blood haemoglobin was determined by an automated cell counter Brazil).
The following covariates were collected and grouped into: socioeconomic -maternal age (< 19 or ≥ 19 years), maternal education (< 9 or ≥ 9 years), maternal skin colour (brown, black, white, or indigenous/yellow), living with a partner (no or yes), head of the family (participant or others), maternal occupation (unpaid or paid job), receipt of the Bolsa Família conditional cash transfer program (no or yes), ownership of household assets (no or yes); environmental -water supply (general water supply, water well or river/rain), sanitary facility (septic tank, rudimentary tank or open air/ river), number of residents in the household (≤ 2, 3, 4 or ≥ 5), type of household (masonry or wood/mix [masonry + wood]), area of residence (rural or urban); obstetric history -menarche age (≤ 13 or ≥ 14 years), history of foetal loss (no or yes), primigravidae (no or yes); and gestational characteristics -smoking during pregnancy (no or yes), gestational urinary tract infection (no or yes), nutritional supplement use (none, iron + folic acid, or multiple micronutrients with VA), number of antenatal care appointments (< 6, 6-8, or ≥ 9), gestational malaria (no or yes), and gestational age at delivery (weeks).
In order to assess the socioeconomic status of the participant's household, a wealth index was created using principal component analysis, according to the presence of household assets 18

Sub-sample analysis
Among participants recruited at the maternity ward, a sub-sample of pregnant women (n = 528) who were attending public antenatal care clinics in the urban area of the municipality were followed-up since the antenatal period. They were recruited for the study prior 20 weeks of pregnancy based on their last menstrual period, and invited for two clinical assessments at [16][17][18][19][20] weeks and at about 28 weeks of pregnancy. For confirmation of gestational age according to a standardised protocol 14 , ultrasound measurements were obtained with a portable SonoSite TITAN ® (SonoSite, Inc., Bothell, WA, USA) by two trained physicians. All images were reviewed by an expert obstetrician not involved in the fieldwork.
Blood samples were collected in both assessment during the antenatal period and shipped to analysis according to previous protocols used in similar field conditions 19 . Serum concentrations of retinol were measured using HPLC methods (HP-1100 HPLC system, Hewlett Packard, Palo Alto, California, USA) 20 , and the values categorized following WHO recommendations (< 0.7 µmol/L -deficiency; 0.7-1.05 µmol/L -insufficiency; ≥ 1.05 µmol/L -sufficiency) 3 . We further explored the VA status by creating two variables deeming the occurrence of VAD and VA insufficiency in both antenatal assessments, regardless if they occurred in any assessment or both: combined VA status -deficiency (not deficient during pregnancy or deficient during pregnancy) and combined VA status -insufficiency (sufficient during pregnancy or insufficient during pregnancy). We also explored the variation of serum retinol concentrations between assessments (serum retinol in the 2 nd assessment minus serum retinol in the 1 st assessment) as 'tertiles of the difference of serum retinol'. Plasma ferritin concentrations were measured by enzyme immunoassays (Ramco, Houston, TX) and the cut off adopted for iron deficiency was < 15 µg/L 2 . Plasma C-reactive protein (CRP, mg/L) was measured by an IMMAGE Immunochemistry System (Beckman Coulter, Brea, CA, USA), adopting concentrations ≥ 5 mg/L as acute inflammation 21 . Plasma ferritin and CRP were only available in the 2 nd assessment (n = 464). Intra-and inter-assay CVs were < 7%.

Statistical analysis
Frequencies were used to characterise the sample, and the Pearson's and Wald's chisquare tests were used to analyse the differences between the categorical variables investigated with the outcomes. The same approach was used for the sub-sample analysis.
Multiple Poisson regression models with robust variance through a hierarchical selection of covariates were used 22 . A hierarchical conceptual model considered four levels of determination, as follows: 1 -socioeconomic; 2 -environmental; 3 -obstetric history; 4gestational characteristics. Covariates associated with each outcome at p ≤ 0.20 in crude analysis, or in view of their significance in the literature, were selected to multiple models. At each level of determination, covariates associated with outcome at p < 0.10 or if conceptually relevant were retained for the final multiple model. Missing observation (<5%) were included in the models by creating missing-value categories. Associations with the outcomes are presented with unadjusted and adjusted prevalence ratios (aPR) and respective 95% confidence intervals (95% CI). All p-values were derived from two-tailed tests. Stata 15 (StataCorp,College Station,Texas,USA) was used to perform the analysis.

Population and characteristics
During the recruitment period, 1,865 hospitalizations of women living in Cruzeiro do Sul were registered, of which 128 were not eligible (16 stillbirths and 112 abortions). The remaining 1,737 women were admitted for delivery; of these 1,538 gave consent and were enrolled in the study. Out of 1,538 postpartum women who agreed to participate in the study, 1,525 (99.2%) had data on GXN, and 1,445 (94.0%) had data on maternal anaemia.
In Table 1, socio-demographic and environmental characteristics of participants are shown. About 20% of parturients were adolescents (< 19 years) and more than 40% of them had less than 9 years of formal schooling. More than three quarters of postpartum women reported 'brown' as their skin colour, with only 11.7% reporting 'white' (data not shown in Tables). The obstetrics and gestational characteristics of participants are given in Table 2.
The majority of participants took supplements during pregnancy (90.5%) and attended more than six antenatal care visits (72.4%).

Prevalence of the outcomes and association with nutrient biomarkers in pregnancy
The prevalence of GXN was 11.5% (95%  and maternal anaemia was detected in 39.4% (95% ) of participants at delivery ( In the sub-sample analysis, VA status in the 1 st assessment and the combined VAD in the antenatal period were associated significantly with GXN. In addition, VA status in the 1 st assessment, as well as combined VAD, combined VA insufficiency, and iron deficiency were associated with maternal anaemia (Table S1). The occurrence of acute inflammation was only 0.2% (data not shown in Tables).
Factors associated with GXN and maternal anaemia Table 3 presents the factors associated with GXN. After controlling for potential confounders, parturients who were living in a household with four or more residents presented a higher prevalence rate of GXN when compared with those sharing the household with fewer members (five or more -aPR 2.06, 95% CI 1. 24-3.41). Smoking during pregnancy was associated with an increased prevalence rate of GXN (aPR 1.78, 95% CI 1. 15-2.78) in comparison with non-smokers. Participants who completed insufficient antenatal care appointments (<6) had higher prevalence rate of GXN when compared with those who had more than 9 appointments (aPR 1.61, 95% CI 1.08-2.40) ( Table 3).
Factors associated with maternal anaemia are presented in Table 4. After adjustment, teenager postpartum women were more likely to be anaemic at delivery than adult parturients (aPR 1.18, 95% CI 1.01-1.38). An inverse gradient was seen for the number of antenatal care appointments, as a higher prevalence rate of anaemia was observed among women who completed insufficient visits (<6: aPR 1.40, 95% CI 1. 15-1.70). Malaria occurrence during pregnancy was positively associated with maternal anaemia in this population (aPR 1.22, 95% CI 1.01-1.49). Compared with multiple micronutrients use, not taking any nutritional supplement during pregnancy was associated with higher prevalence of maternal anaemia (aPR 1.27, 95% CI 1.01-1.62), whereas the use of iron-folic acid supplement was not significantly associated (Table 4). Further adjustment for gestational age at delivery did not change the associations observed for both outcomes.

Main findings
High prevalence rates of GXN and maternal anaemia were found in this populationbased study in Western Brazilian Amazon. The prevalence of GXN (11.54%) was more than twice the WHO cut-off for public health significance in pregnancy 3 . The prevalence of maternal anaemia affected almost 40% of the participants. GXN was associated with VAD during pregnancy, regardless of the time of assessment, as well as with the number of residents living in the same household, smoking during pregnancy, and the insufficient number of antenatal care appointments. Maternal anaemia was associated with almost all biomarkers of VA and iron status measured in the sub-sample analysis, and with maternal age, the insufficient number of antenatal care appointments, malaria episode and nutritional supplement use in pregnancy.

Strengths and limitations
The population basis of our study allowed us to obtain estimates of prevalence rates that are representative of this low-resource study area. However, even though the interview used for assessment of GXN is a standardised instrument proposed by WHO, and adequate for epidemiological purposes, the method used relies on the respondents' memory recall and ability to understand the questions. Although associations of GXN with VAD in pregnancy were confirmed regardless of the time of assessment in our sub-sample analysis, a recall bias cannot be discharged. The lack of a local term to describe the symptom may also have hindered comprehension 7 .
Caution is needed in the interpretation of our results considering the limitations of the tool used to assess GXN. Historically, the assessment of night blindness by the WHO questionnaire has been one of the most commonly used and recommended methods for assessing VAD in vulnerable populations, even though the reported tool does not correlate strongly with serum retinol 7,23 , likely owing to the hepatic regulation of this biomarker when retinol stores are replete in the liver 24 . Nevertheless, this is a low-cost, less-invasive method to monitor VA status in populations.

Interpretation
Global estimates of night blindness in women of childbearing age, retrieved from the WHO Global Database on Vitamin A Deficiency 25 , reveal important prevalence among some selected countries, especially in Asia, although the last update occurred more than 10 years ago. Prevalence rates of less than 10% were registered in Indonesia (1.7%), Madagascar (7.5%), and Nigeria (7.7%), whereas high prevalence rates were observed in Bolivia (14.0%), Nepal (19.6%), and in the district of Medak, India (35.0%) 25 . There are no countrywide data for GXN in Brazil; most investigations have been concentrated in Rio de Janeiro, where the prevalence of GXN of 9.9% was found in a maternity hospital 8 . This result aligns with our findings in Brazilian Amazon. Moreover, the last Demographic Health Survey conducted in Brazil showed that 12.3% of women of childbearing age were VA deficient 9 .
Poor social, environmental, and pregnancy-related factors conditions have been associated with GXN 3,26 . In rural Nepal, a population-based case-control study found that patients with GXN were more likely to be illiterate, to be from lower castes, and to live in households of poor quality 26 . Similarly, in a population-based trial of newborn VA supplementation in rural India, having a concrete roof, being literate, and being pregnant during adulthood lowered the odds of GXN, whereas higher parity increased the odds 10 . A cross-sectional study in a maternity ward in Rio de Janeiro city found that living in wealthier areas of the city and receiving antenatal nutritional assistance reduced the odds for GXN, whereas having a history of abortion and having anaemia during pregnancy increased the odds 8 . Counselling during antenatal care may act on the improvement of lifestyle behaviours, like quitting smoking and increasing the intake of nutrient-rich foods, especially in VA.
Pregnant women who live in a household with a high number of residents are likely to share their home resources, lowering food availability to supply their own physiological needs during pregnancy.
The WHO global estimates indicate that anaemia in pregnancy is a worldwide issue, and almost all countries had prevalence rates higher than 20% in 2011 27 . In this report, estimates for Brazil set anaemia in pregnancy as a moderate public health problem, with a prevalence rate of 32% (95% CI 11-62) and mean haemoglobin of 116.0 g/L (95% CI 106-128). In our study, prevalence of anaemia in parturients was even higher than those estimated by the WHO for Brazil, comparable with rates for Asia 27 , where gestational malaria can be also one of the associated factors with maternal anaemia.
Classical factors associated with anaemia include iron deficiency, due to low consumption, higher needs or inadequate absorption, other micronutrient deficiencies (e.g. folate and VA), infections (e.g. malaria and HIV), and haemoglobinopathies 19,27 . The ironfolic acid supplementation is a recognized intervention to reduce the anaemia burden in pregnancy 4,28 . In our study, anaemia was associated with being a teenage parturient, presenting malaria episodes, attending insufficient antenatal care visits, and not using nutritional supplements in pregnancy. Nutritional demands are higher for teenagers than for adult women as they are still undergoing physiological development, and pregnancy in adolescence exacerbates such demands by concomitantly needs for the foetus growth 10 .
Malaria is a proven risk factor for anaemia in pregnancy, because the rupture of red blood cells during Plasmodium parasite's metabolism lowers the red blood cell count 29 . During antenatal care, interventions aimed to prevent anaemia are advised, such as encouraging on intake of iron-rich foods and iron-folic acid supplementation 4 .
Noteworthy, such high prevalence of GXN and maternal anaemia in this study ascertain the importance of achieving a good nutritional status and health assistance during pregnancy in a context of social and environmental vulnerability 12,29 . The current nutrition strategies recommended by the WHO for targeting micronutrient deficiencies in pregnancy include supplementation with only iron and folic acid. Recently, multiple micronutrients supplementation has been proven effective to prevent of harmful effects of micronutrient deficiencies in pregnancy 30 . Taking into account the positive results for multiple micronutrients and the synergy among micronutrients 31 , the WHO nutrition-related protocols for antenatal care should be reviewed.

Conclusion
GXN and maternal anaemia were identified as significant public health problems in the Brazilian Amazon municipality studied. Such high prevalence of GXN and maternal anaemia may reflect the poor nutritional status experienced by these women since pregnancy, which can have deleterious lifetime effects for the dyad mother-infant. Sociodemographic, environmental and gestational characteristics were associated with the occurrence of GXN and maternal anaemia. Most of these factors can be addressed during antenatal care, underlying the importance of rethinking current protocols related to nutrition in pregnancy.    * Haemoglobin at delivery < 110 g/L; ** Gestational night blindness; *** Unadjusted prevalence ratio with respective 95% confidence interval; **** Adjusted prevalence ratio with respective 95% confidence interval. 's exact test; *** Between 16 to 20 weeks of pregnancy; **** About 28 weeks of pregnancy; **** For 'insufficient during pregnancy' was considered insufficiency (serum retinol < 1.05 µmol/L) in at least one assessment or both; for 'deficient during pregnancy' was considered deficiency (serum retinol < 0.7 µmol/L) in at least one assessment or both; ***** Totals differ from the total number of participants studied because of missing values.   (Yang et al., 2016;Chelchowska et al., 2011). Também em linha com outras investigações, porém positivamente, a sazonalidade no momento em que as amostras sanguíneas foram obtidas mostrou-se um bom preditor das concentrações de retinol, tendo em mente que isso ocorra possivelmente pela maior oferta de certos alimentos em algumas épocas do ano. As prevalências de XN gestacional e de anemia no parto foram de alarmantes proporções na análise transversal de base populacional (Artigo 3). Apesar das limitações do método utilizado para avaliação da ocorrência de XN na gestação e de se recomendar cautela em sua interpretação, a prevalência encontrada foi maior que o dobro ao ponto de corte adotado pela OMS como indicador de um problema de saúde pública associado à DVA em uma população.