1. Introduction

Iron is a trace element in the body with an amount between 4 and 5 g in the heminic or non-heminic form. In organism, it is one of the constituents in hemoglobin and myoglobin which intervene in transport of gases. This micronutrient also plays a central role in a series of enzymatic reactions implicated in oxydoreduction metabolism for energy production and is essential for cellular respiration ^[1]. In addition, iron contributes to physical performance, cognitive and resistance to infection ^{[2, 3]}. According to international institutions such as World Health Organization and United Nations Children's Fund, 4 to 5 billion of population are affected by iron deficiency worldwide ^[4]. This iron deficiency is mainly due to imbalance between intake and iron requirements of the body. Thus, iron appeared, among all the micronutrients, like one of those whose coverage needs poses in human species, most practical problems to solve. Furthermore, physiological state of pregnancy with high iron requirements changes iron status of pregnant women ^{[5, 6, 7, 8]}. Some factors such as infectious and inflammatory syndromes degrade iron metabolism in populations of developing countries especially among women of reproductive age ^{[9, 10]}.

Among these factors, human immunodeficiency virus and antiretroviral treatment would lead to alteration of the different metabolic pathways of iron ^{[11, 12]}. Recent works in developing countries have shown changes of iron metabolism in women during pregnancy on antiretroviral therapy ^{[13, 14]}. In Côte d’Ivoire, very few investigations have reported alteration of iron status in HIV infected pregnant women with antiretroviral therapy. This study aims to evaluate and characterize interaction of pregnancy, HIV and antiretroviral therapy on iron metabolism in women of reproductive age during pregnancy in Abidjan. In this context, three groups of women at different trimesters of pregnancy were selected specifically to achieve the following objectives:

•  Compare depending on the trimester the biological assessment of iron status among the three groups of women;

•  Report and compare different proportions of the main biological parameters of iron metabolism during the three trimesters of pregnancy and between the three groups of women;

•  Identify and compare the different prevalences of iron status components according to trimesters between the three groups of women;

•  Reveal the group of pregnant women most exposed to possible alteration of iron metabolism;

•  Determine stage of pregnancy during which interaction of pregnancy, HIV and antiretroviral therapy significantly affects iron status of women.

2. Materials and Methods

The investigations were cross-sectional and descriptive study which took place from 21 October 2009 to 21 December 2012 in the Integrated Centre for Bioclinical Research of Abidjan (ICBRA). The study involved 405 women aged from 18 to 45 years with mean age of 27.1 ± 0.6 years during pregnancy. This sample of pregnant women consists of 130 non HIV infected pregnant women as control, 135 HIV infected pregnant subjects without antiretroviral treatment (ARVT) and 140 HIV infected women receiving antiretroviral therapy (ARVT). These pregnant women came for consultation in Integrated Centre for Bioclinical Research of Abidjan (ICBRA) for prenatal examinations and biological monitoring of their HIV infection for some. Control pregnant women were composed of 40 subjects in the first trimester, 40 in the second trimester and 50 subjects in the last trimester (Figure 1). Women infected with HIV and without antiretroviral therapy are composed of 50 subjects in the first trimester, 45 subjects in the second trimester and 40 women in the last trimester. For HIV infected pregnant women with antiretroviral treatment, 40 women were in first trimester, 45 subjects in second trimester and 55 women in third trimester of pregnancy. In addition, The two group of infected pregnant women included 99 % of HIV-1 against 1 % HIV-2. Tritherapy most widely prescribed to pregnant women is characterized by Zidovudine (AZT), Lamivudine (3TC) and Nevirapine (NVP). This set of women from different municipalities and suburbs of Abidjan (Côte d'Ivoire) was selected after informed consent of each woman on the objectives of the investigation. The 405 selected pregnant women have not presented major complications of hypertension, Diabetes, rheumatoid arthritis. In contrast, those with recently reported major health concerns that is transfused, indicating digestive and gynecological diseases were excluded.

Figure 1. Sampling of study population according to selected sites and different trimesters of pregnancy

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The mean age of enrolled women in the study was 25.2 ± 0.4 years, 25.3 ± 0.5 years and 30.8 ± 0.9 years respectively in control subjects, HIV infected subjects without antiretroviral treatment and HIV infected women with antiretroviral treatment. HIV infected without antiretroviral therapy subjects included more adolescents (28.5 %) compared with controls (8.5 %) and women receiving antiretroviral therapy (2 %). These had generally a body mass index (BMI) of 23.5 ± 0.3 kg.m^-2, 23.9 ± 0.4 kg.m^-2 and 24.6 ± 0.6 kg.m^-2 respectively in controls, HIV infected pregnant women without ARVT and HIV infected pregnant women on ARVT. Weight status was abnormal (underweight and overweight) in 32.3 % of controls against 47.4 % et 46 % in HIV infected women.

The gravidity and parity had mean values of 12.7 ± 0.1 and 1.1 ± 0.1; of 3 ± 0.1 and 1.4 ± 0.1; of 1.8 ± 0.2 and1.8 ± 0.2 respectively in controls, HIV pregnant women without ARVT and HIV infected pregnant women. Several study subjects (100; 106 and 84 respectively) had indicated a pregnancy for their obstetric histories. In this same way, respectively 52.3 %; 62.2 % and 94 % of women selected had at least one childbirth. In terms of space between births, 63.8 % of control women against 67.4 % 56 % of infected women have observed less than 3 years between pregnancies. Study population also included married, widows, single and subjects living in concubinage (Table 1).

Table 1. General characteristics of the study population

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For each of the recruited women, a blood sample collected in dry tubes and tubes with 5 ml anticoagulant each were performed fasting bend of the elbow in the morning. Whole blood collected in tubes with anticoagulant (EDTA) achieved the CD4 counts by flow cytometry with Fascalibur® and the blood count by the Sysmex PLC Xt 2000i. The collected blood in dry tubes was centrifuged at 3000 tours for 5 minutes to obtain the serum. The obtained serum allowed to determine HIV status and biochemical data assessment of iron status. For HIV serology, the method most used in the care centers. is the use of two successive tests. Once the first test (Determine) is positive, we proceed to discrimination test (Genie II HIV-1/HIV-2) to determine the type of HIV. The quantitative determination of biochemical parameters (serum iron. serum transferrin and serum ferritin) in human serum is based on a colorimetric technique available on automated COBAS INTEGRA 400. For this determination, the COBAS INTEGRA kits Iron (IRON). Tina-quant Transferrin ver.2 (TRSF2) TRSF2 test and Ferritin Gen.2 ID 0-567 test (FERR2), ID 0-567 test containing in vitro diagnostic reagents were used. The total iron binding capacity (TIBC) and the saturation coefficient of transferrin (SCT) were obtained by calculations.

To better appreciate the parameters of biological assays, conventional criteria were selected. They associated the recommendations of international organizations (WHO), French Society of Clinical Biology (SFBC/France), French Society of Hematology (SFH/France-Group of Cellular Haematology), Society of Nutrition and Diet of the French Language (France). Centre for Disease Control and Prevention (WHO/CDCP) and Institute of Medicine ^{[15, 16, 17, 18]}.

The obtained values of the biological parameters of iron status were subjected to a factorial analysis of variance (ANOVA) with one factor (the three groups of women) in order to evaluate in all their evolution. These statistics treatments with Statistica Statsoft program Windows version 7.1 were associated NEWMAN-KEULS as multivariate test post hoc to specify the probable groups of women significantly different ^[19]. An interaction of HIV, antiretroviral therapy and the three trimesters of pregnancy on biological parameters of iron metabolism assessment were performed by multiple analysis of variance (AMOVA).

Different observed proportions of biological indicators of iron status were compared by the G test or log Likelihood ratio test. Test G and AMOVA were carried out with the software R.2.0.1 Windows version ^[20]. The level of significance was defined for a p value < 0.05.

Experimental procedures and protocols used in this study were approved by ethical committee of Health Sciences, Nangui Abrogoua University. These guide lines were in accordance with the internationally accepted principles for laboratory use and care. Approval was also obtained from the Ministry of Higher Education and Scientific Research and the Ministry of Health and Public Hygiene in the Republic of Côte d'Ivoire.

3. Results

The results of study showed that significant differences between pregnant women group in MCV, serum iron, TIBC and serum ferritin (Table 2). Thus, infected pregnant women on antiretroviral therapy have recorded high values of these biological parameters except for TIBC compared with other women group of study. Women which were not HIV infection, reported values of these significant biological parameters lower than other women in this study.

Table 2. Mean values of iron status biological parameters in the three pregnant women

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Table 3. Variation of iron status parameters depending on different women groups during pregnancy

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In Table 3, a significant interaction of the three factors like, pregnancy, HIV and antiretroviral therapy was observed on all biological parameters exception with saturation coefficient of transferrin. For these biological parameters, infected pregnant women on antiretroviral therapy have indicated low values of red blood cells, hemoglobin, MCHC, serum transferrin, and TIBC to the first trimester of pregnancy. However, serum ferritin was high in these women compared with other women of study. Iron stores were significantly decreased in control women and infected pregnant women without ARVT throughout the pregnancy. To the second and third trimesters of pregnancy, infected women without antiretroviral therapy have revealed altered hemoglobin, hematocrit, MCV, serum transferrin and TIBC compared with other two groups of women.

Results in Table 4 showed that controls have indicated high prevalence of microcytosis compared with other women. However, HIV infected pregnant women on antiretroviral therapy have reported macrocytosis prevalence higher than controls. In addition, HIV infected pregnant women without antiretroviral therapy have not concerned by macrocytosis. Moreover, prevalences of anaemia were significantly not different between the three groups of women. But, HIV infected pregnant women were more anaemic compared with other women of study.

For biochemical parameters, control women have revealed high proportion of low values of iron and ferrition compared with HIV infected pregnant women with and without antiretroviral therapy. Moreover, all HIV infected pregnant women have recorded normal values of ferritin. In contrast, HIV infected pregnant women have presented increased proportions of high values of serum ferritin. However, no control and no woman without antiretroviral therapy have revealed ferritin values above normal limit. Furthermore, controls have recorded large proportions of high and low values of serum transferrin and TIBC.

Table 4. Proportions of main biological parameters of iron status in all study subjects

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In Table 5, antiretroviral therapy, HIV and pregnancy have an impact on anaemia at first trimester of pregnancy. HIV infected pregnant women with were anaemic at this stage of pregnancy compared with other women of study. Microcytosis and hypochromia were impacted by the three factors throughout the three trimesters of pregnancy. Thus, control pregnant women have recorded more microcytosis and hypochromia during first and third of pregnancy compared with HIV infected women. But, HIV infected women on antiretroviral therapy have presented prevalences of microcytosis (62.2 %) and hypochromia (68.9 %) higher than other study subjects. In the same way, types of anaemias depending on erythrocyte parameters’ variation were impacted by HIV, pregnancy and antiretroviral therapy. Indeed, HIV infected pregnant women with antiretroviral therapy have revealed throughout the pregnancy large prevalences of normochromic normocytic anaemia and hypochromic normocytic anaemia at the two last trimesters of pregnancy. In addition, hypochromia microcytic anaemia was reported in all controls at first trimester of pregnancy. This anaemia was more observed in all HIV infected pregnant during the second and third trimesters of pregnancy. In these women, hypochromic normocytic anaemia and normochromic microcytic anaemia were recorded at beginning of pregnancy. Furthermore, these two types of anaemia were not observed in controls and infected women without antiretroviral therapy at the two last trimesters of pregnancy (Table 5).

Table 5. Compared proportions of erythrocyte parameters depending on different trimesters of pregnancy between three groups of women

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The proportions of biochemical parameters according to their limit values were affected by HIV, pregnancy and antiretroviral therapy. In this way, control women were low in proportion with values of iron below normal during second and third trimesters of pregnancy. These women were in third trimesters with iron values above limit. The same observation was reported in these women with serum transferrin and TIBC. Women with antiretroviral therapy have revealed large proportions of saturation coefficient of transferrin below and above reference value. In addition, only these subjects have presented values of ferritin above normal throughout the pregnancy (Table 6). In contrast, several controls have recorded values of ferritin below reference value compared with other women of study.

The finding in Table 7 showed that few HIV infected pregnant women with ARVT have reported normal iron status (8 %). There were several HIV infected pregnant women without antiretroviral therapy which indicated normal iron status (37.1 %) compared with other women of study. The observed components of iron status in this were iron deficiency (ID), iron deficiency anaemia (IDA), inflammatory anaemia (IA) and inflammatory anaemia associated with iron deficiency (IA+ID). ID an IDA were not observed in HIV infected pregnant without antiretroviral therapy. These types of iron status components were largely indicated in control pregnant women. IA was more recorded in HIV infected pregnant on antiretroviral therapy. However, HIV infected pregnant without antiretroviral therapy have more reported IA+ID. In this investigation, only controls have indicated all observed components of iron status.

Table 6. Distribution of proportions of biochemical iron status parameters according to different trimesters of pregnancy between the three groups

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Table 7. Distribution of iron status components according to three pregnant women group

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In Table 8, all components of iron status were affected by HIV, antiretroviral therapy and throughout the three trimesters of pregnancy. Thus, at first trimester of pregnant, all HIV infected women with antiretroviral therapy have reported an abnormal iron status compared with other women. After, there were respectively HIV infected women without ARVT in second trimester and controls in third trimesters of pregnancy which have recorded an abnormal iron status. In this same way, control women were concerned by IDA throughout the pregnancy. IA was more observed during the three trimesters of pregnancy in HIV infected with antiretroviral therapy and those without antiretroviral therapy were affected IA+ID (Table 8).

Table 8. Changes of iron status components during pregnancy in pregnant women group

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4. Discussion

Iron metabolism during pregnancy is perpetually disrupted for several reasons. In this study, all biological parameters of iron status assessment are strongly impaired in all selected pregnant women. Indeed, haematological parameters were influenced by the interaction of the three trimesters of pregnancy, HIV and antiretroviral therapy in all study pregnant women. This led to the strong prevalences of microcytosis, macrocytosis and hypochromia. Anaemia is present in all pregnant women with high prevalences of 65.5 %, 62.2 % and 71.4 % respectively in control pregnant women, HIV infected pregnant women without antiretroviral therapy and HIV infected pregnant women on antiretroviral treatment. These prevalences are sufficiently high compared to work in Nigeria in HIV infected pregnant women ^[21]. These authors reported a prevalence of 49.3 % of anemia in these pregnant women. According to dynamic concept of anaemia, it occurs only with degradation of plasma components and iron stores. For this investigation in pregnant women, biochemical indicators for evaluating metabolic pathways of iron, have a real influence of combined effect of pregnancy, HIV and antiretroviral therapy. In addition, iron stores in these pregnant women are disrupted. Several factors explain alteration of iron stores in pregnant women.

Pregnancy is a physiological condition which requires a strong demand of iron to deal with different needs of the mother and fetus. A quantity of 1290 mg of iron is recommended to overcome increase in plasma volume and red cell mass, synthesis of new red blood cells, formation of the placenta, fetal growth and blood loss during delivery ^[22].

This quantity of iron is given to organism of pregnant woman by supplementation or through diet. In developing countries, food for population in general and especially in pregnant women is very poor in micronutrients. According to some studies, women during pregnancy have a diet highly devoid of iron easily bioavailable ^{[23, 24]}. Other investigations reported an insufficiency of protein (total protein and albumin) in the body women during pregnancy in southeast Nigeria ^[25]. In our study, 82 % of apparently healthy pregnant women considered as controls have a depletion of iron. This has resulted in these pregnant women to large prevalences of hypochromic and microcytic anaemia with 100 %, 90 % and 100 % respectively in the first, second and third trimesters of pregnancy. This iron depletion has led to a high prevalence of iron deficiency anaemia significantly observed in control pregnant women. This result is similar to those which were reported high rates of anaemia among women of reproductive age, pregnant and non-pregnant women in Abidjan ^{[6, 7, 8]}.

The main reasons for this anaemia as also reported in pregnant women in Nigeria were iron deficiency, folates and vitamin B₁₂ ^[7].

In addition, the degradation of iron pathways during pregnancy is due to parasitosis, infectious and inflammatory syndromes [12-26]^[12]. Two groups of women in our study are pregnant and HIV infected. This human immunodeficiency virus causes infectious and inflammatory syndrome ^[27]. This explains the disruption of iron stores in these study subjects which have remained apparently normal. All HIV infected pregnant women show during the three trimesters of pregnancy normal levels of serum ferritin (iron stores). This observation in iron stores leads to increased prevalence of normocytic normochromic anaemia, hypochromic normocytic anaemia and even normochromic microcytic anaemia in these infected pregnant women. This is caused by alteration of all biochemical indicators of iron status assessment.

This alteration of different plasma components and iron stores among all pregnant women in our study is also associated with iron deficiency, iron deficiency anaemia, inflammatory anaemia and inflammatory anaemia with iron deficiency. This result was also recorded in HIV-infected African women ^[28].

Action associating HIV inflammation leads to significant prevalence of inflammatory anaemia associated with iron deficiency. Several factors are involved in pathophysiology of inflammatory anaemia which is related first and foremost to an insufficiency of erythropoiesis resulting from inhibition of erythroid progenitors and disruption of synthesis and action of erythropoietin. These perturbations are induced by cytokines whose secretion is increased in the inflammatory process (interleukin-1, TNF-alpha, interferon-gamma) by directly inhibiting expression of erythropoietin gene ^[29]. A disorder of iron metabolism also would intervene. During inflammatory process, macrophages sequestrate iron freed by hemolysis. This difficulty in iron mobilization from reserves leads to a decrease in the synthesis of hemoglobin, in which an increase in mitoses number is responsible microcytosis. The decrease of plasma transferrin (iron transport protein) is linked on one hand to its hypercatabolism in the inflammatory focus, on the other hand the reduction of its synthesis (iron stores are full, as evidenced by the normal or high ferritin). Finally, a moderate shortening of lifespan of red blood cells (hemolysis) is observed ^{[30, 31]}. These same observations were reported in some infected subjects with HIV ^[32]. Among these subjects with anaemia, correlation has reported between parameters of iron metabolism such as serum iron, serum ferritin and soluble transferrin receptor and interferon-gamma (IFN-gamma). The observed change of erythropoietin in these same subjects has indicated a dysfunction in bone marrow. In addition, all these changes of iron metabolism during inflammation are the consequence of the hepcidin effect. In fact, the secretion of cytokines during inflammation activates a protein recently identified hepcidin. This protein prevents the export of iron from cells duodenal and reticuloendothelial system, but not measured in practice ^[33]. This could justify the high rates of inflammatory anaemia and this anaemia associated with iron deficiency in HIV infected pregnant women with and without antiretroviral therapy. Iron deficiency among pregnant women infected with HIV is real. Besides inflammatory anaemia observed with a high prevalence, iron deficiency anemia is also reported in women with rates between 8 and 10 % depending on the trimester. These observed prévalences of iron deficiency anaemia, however, are below that revealed in 136 HIV infected pregnant women corresponding to 14.7 % ^[34]. Iron is not the only micronutrient whose metabolism is altered during HIV infection in pregnancy. Other micronutrients such as folates, zinc, copper and vitamin B series are affected by their degradation pathway in HIV infection during pregnancy ^[35]. In addition, findings have revealed that during antiretroviral therapy in women during pregnancy and HIV infection, degradation of iron metabolism parameters assessment, is generally observed. Similar results were indicated in HIV infected pregnant women with high antiretroviral therapy in Latin America and caraïbes, with a prevalence of anaemia of 12.2 % against 62.2 % (Pregnant Women without antiretroviral therapy) and 71.4 % (pregnant women on antiretroviral therapy) ^[36]. In the event of our investigation, prevalence of anemia is higher than that indicated in the Latin American and caraïbean. Antiretroviral therapy depresses prevalence of opportunistic gastrointestinal diseases and severe gastroenteritis. These pathologies affect the absorption of micronutrients ^[37]. However, several medications against HIV (NRTI3) can inhibit the replication of mitochondrial DNA and cause vomiting and diarrhea that may reduce absorption or increase losses of several micronutrients. This could also explain the degradation of iron metabolism in women in our study. Nutrition and pharmacology of antiretrovirals are actually related as this study so well indicates ^{[38, 39, 40]}.

5. Conclusion

The performed study in HIV infected women during pregnancy indicate that all biological indicators iron metabolism of evaluation are degraded in all the subjects selected. In addition, this investigation reveals that the interaction of pregnancy, HIV and antiretroviral therapy was observed in all pregnant women selected for most biological parameters. This alteration leads to significant higher rates of various components of iron status. In this way, prevalence of inflammatory anaemia and anemia of inflammatory anaemia associated with iron deficiency are more important. This observation is critical for infected pregnant women with antiretroviral therapy. However, HIV infected women without antiretroviral therapy reported a high prevalence of inflammatory anaemia associated with iron deficiency. Moreover, low rates of iron deficiency and iron deficiency anaemia were observed in enrolled pregnant women. Degradation of biological indicators assessment of iron metabolism is justified by the high need for iron and hemodilution during pregnancy, through the action of HIV on erythropoietin and antiretroviral medication effect on iron absorption. In this context, a measure of certain parameters of iron metabolism such as soluble transferrin receptor and hepcidin should be considered for better estimate iron stores of women during pregnancy with HIV infection.

Acknowledgement

The authors are grateful to all laboratory managers and staff of Integrated Centre for Bioclinical Research of Abidjan/Côte d’Ivoire for their support during our investigations. Special thanks are also addressed to Dr. Léonie Clémence KOUONON for her guidance during drafting of this study. We thank Dr. Ignace Kouadio Kouassi for his assistance in building the database and planning data analysis.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.

Authors’ Contributions

All authors contributed equally in the study. They made substantial contributions to the design of the study, the collection of the data as well as the preparation and analysis of the data. They also drafted the manuscript and gave final approval for its submission to the journal for consideration of publication.

Declaration of Interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

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