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“PARAFRASE JURNAL”
Diajukan untuk memenuhi Tugas Ulangan Tengah Semester (UTS) pada
matakuliah Metodologi Penelitian dan Biostatistik

Dosen Penguji : Sulistyaningsih, S,KM., MH.Kes

DISUSUN OLEH
Nama : Mirna Apriliani
Nim : 1710104329
Kelas : 7 F

PROGRAM STUDI DIV BIDAN PENDIDIK

FAKULTAS ILMU KESEHATAN
UNIVERSITAS ‘AISYIYAH
YOGYAKARTA
TAHUN 2017
A. IDENTITAS JURNAL
1. Nama Jurnal : The American Journal of Clinical Nutrition
2. Nama Artikel : IRON DEFICIENCY AND ANEMIA ARE
PREVALENT IN WOMEN WITH MULTIPLE
GESTATIONS (Defisiensi Besi Dan Anemia Terhadap
Wanita Dengan Gestasi Multiple)
3. Publisher : American Society for Clinical Nutrition, Inc.
4. H-Index : 283
5. Quartil : Q1
6. Websit : http://ajcn.nutrition.org/content/104/4/1052.full.pdf+html

B. HASIL PARAFRASE
1. Abstrak
Anemia dan zat besi selama kehamilan telah dikaitkan dengan
peningkatan risiko pada saat persalinan dan pengaru pada bayi yang
dikandungnya seperti kelahiran prematur, berat lahir rendah (BBLR), dan
rendahnya zat besi neonatal saat lahir. Penelitian ini bertujuan untuk
mengetahui defisiensi besi dan anemia terhadap perempuan pada wanita
dengan gestasi multiple. Desain penelitian mengunakan observasional
analitik dengan metode analisi data secara cross sectionally. Instrumen
yang digunakan adalah kuesioner. Sampel penelitian sebanyak 83 ibu hamil
dengan gestasional multiple (umur 20-46 tahun) direkrut dari 2011 hingga
2014. Analisis statistik meggunakan uji Shapiro-Wilk dan dilakukan
perbedaan statistik dengan menggunakan Wilcoxon's uji rank-sum. Hasil
uji statistik dianggap signifikan di P < 0,05. Semua analisis statistik
dilakukan dengan penggunaan perangkat lunak JMP 10.0 (SAS Institute
Inc.)
Hasil penelitian diperoleh prevalensi pada iron deficiency meningkat
secara signifikan dari kehamilan sampai persalinan sebesar 9,6%. Ibu hamil
yang persediaan besi serum feritin habis selama kehamilan akan memiliki
risiko anemia 2 kali lipat lebih besar saat melahirkan, dan 25%
mengembangkan anemia defisiensi besi (IDA). Secara keseluruhan, 44,6%
dari 83 wanita hamil mengalami anemia saat melahirkan, dan 18% wanita
mengalami penurunan zat besi. Wanita dengan persentil eritropoietin 0,75
selama kehamilan menunjukkan risiko anemia 3 kali lipat lebih besar. Hal
itu menunjukkan bahwa eritropoietin adalah prediktor sensitif anemia saat
melahirkan. simpulan yang didapatkan yaitu ekurangan zat besi dan anemia
 sangat umum terjadi pada wanita dengan beberapa gestasi. Tambahan
skrining dan suplementasi zat besi diperlukan pada populasi berisiko tinggi.
Hal ini mengingat diketahui antara kekurangan zat besi dan anemia pada
ibu dan neonatal tidak sehat.
Kata kunci : anemia, defisiensi besi, multiple births, kehamilan
2. Pendahuluan
a. Latar belakang masalah
Anemia dan zat besi selama kehamilan telah dikaitkan dengan
peningkatan risiko pada saat persalinan dan pengaru pada bayi yang
dikandungnya seperti kelahiran prematur, berat lahir rendah (BBLR),
dan rendahnya zat besi neonatal saat lahir. Hal ini Perlu diperhatikan
karena kekurangan zat besi selama kehamilan tersebut terkait dengan
hasil kehamilan yang merugikan yang diketahui lebih banyak terjadi
pada saat persalinan dan pengarunya pada bayi baru lahir. Namun,
Sedikit perhatian yang telah diberikan pada wanita dengan gestasi
multipel.
b. Tujuan
Untuk mengetahui defisiensi besi dan anemia terhadap perempuan
pada wanita dengan gestasi multiple.
3. Metode
Desain penelitian mengunakan observasional analitik dengan metode
analisi data secara cross sectionally. Instrumen yang digunakan adalah
kuesioner. Sampel penelitian sebanyak 83 ibu hamil dengan gestasional
multiple (umur 20-46 tahun) direkrut dari 2011 hingga 2014. Sampel darah
diperoleh selama kehamilan dan saat melahirkan digunakan untuk menilai
hemoglobin, serum feritin (SF), reseptor transferrin terlarut, hepcidin, besi
serum, eritropoietin, serum folat, vitamin B-12, protein C-reaktif, dan
interleukin. Analisis statistik meggunakan uji Shapiro-Wilk dan dilakukan
perbedaan statistik dengan menggunakan Wilcoxon's uji rank-sum. Data
yang tidak terdistribusi secara normal diubah logaritmik, dan alat geometrik
dihitung Tes t Student dan ANOVA digunakan untuk menguji indikator
besi terdistribusi normal. Hasil uji statistik dianggap signifikan di P < 0,05.
 Semua analisis statistik dilakukan dengan penggunaan perangkat lunak
JMP 10.0 (SAS Institute Inc.)
4. Hasil
Prevalensi pada iron deficiency meningkat secara signifikan dari
kehamilan sampai persalinan sebesar 9,6%. Ibu hamil yang persediaan besi
serum feritin habis selama kehamilan akan memiliki risiko anemia 2 kali
lipat lebih besar saat melahirkan, dan terdapat 25% anemia defisiensi besi
(IDA). Secara keseluruhan, 44,6% dari 83 wanita hamil mengalami anemia
saat melahirkan, dan 18% wanita mengalami penurunan zat besi. Wanita
dengan persentil eritropoietin 0,75 selama kehamilan menunjukkan risiko
anemia 3 kali lipat lebih besar. Hal itu menunjukkan bahwa eritropoietin
adalah prediktor sensitif anemia saat melahirkan.
Mayoritas wanita yang sedang hamil bayi kembar 64% melahirkan
dengan sesar. Prevalensi ini sedikit lebih rendah dari prevalensi nasional
tahun 2008 (75%) pada wanita yang hamil bayi kembar. Dalam penelitian
ini, 18 wanita yang hamil dengan kembar tiga dan ibu yang hamil kembar
empat melalui operasi caesar pengiriman. Persalinan prematur terjadi pada
kohort ini karena hampir 65% wanita dikirim sebelum 37 minggu
kehamilan. Dari wanita yang melahirkan prematur 16,7% memiliki jumlah
PTB <32 minggu kehamilan dan satu ibu hamil dengan kembar tiga sangat
preterm <28 minggu kehamilan. Mayoritas dari neonatus 71,5% yang lahir
dari wanita ini memiliki LBW <2500 gr dan 9,7% dilahirkan dengan sangat
LBW <1500 g). Angka kematian neonatal pada kelompok ini adalah 1,6%
(3/186 kelahiran hidup).
5. Pembahasan
Anemia adalah penurunan jumlah sel darah merah dalam siklus
darah. Anemia juga diartikan sebagai kondisi dimana sel darah merah
menurun sehingga kapasitas oksigen ketubuh organ-organ vital pada ibu
dan janin menjadi berkurang. Baik zat besi dan anemia umum terjadi pada
risiko tinggi pada populasi ibu hamil dan bersalin. Anemia ditunjukkan
pada 45% wanita selama trimester ketiga yang 4 kali lipat lebih tinggi
mengalami anemia dari yang diperkirakan. Meskipun zat besi awal adalah
penentu yang signifikan. Anemia saat melahirkan pada wanita dengan
multiple gestations, namun tidak ada pedoman skrining dini untuk
kelompok ini.
American College of Obstetricians and Gynecologists Practice
Buletin anemia pada kehamilan mengemukakan erythropoietin adalah
hormon yang diproduksi oleh sel-sel khusus di ginjal yang merangsang
sumsum tulang untuk meningkatkan produksi sel darah merah. Hal ini
ditunjukkan menjadi penentu terkuat dari hemoglobin ibu saat melahirkan.
Karena korelasi tinggi ditunjukkan antara eritropoietin selama kehamilan
dan hemoglobin saat melahirkan dan temuannya eritropoietin tidak
terpengaruh oleh peradangan pada ibu. Skrining untuk eritropoietin tinggi
selama kehamilan dapat membantu untuk mengidentifikasi wanita yang
berisiko tinggi mengalami anemia saat melahirkan.
Kekurangan zat besi ibu lazim terjadi pada wanita dengan kehamilan
multipel dan meningkat secara signifikan dari kehamilan sampai persalinan.
Data NHAN (1999-2006) memperkuat penelitian dengan prevalensi zat
besi yang turun secara signifikan lebih tinggi ditunjukkan pada trimester
pertama dan kedua. Ibu dengan gestasi multiple melahirkan bayi mereka
dengan data 65% populasi mengalami prematur. Tingginya prevalensi
anemia dan kurangnya zat besi yang tercatat di pengiriman ditunjukkan
kira-kira 5 minggu lebih awal dari pada istilah tipikal usia kehamilan (40
minggu). Anemia diketahui dapat meningkatkan risiko premature dan
BBLR. Hal ini merupakan hasil kelahiran buruk yang sangat tinggi pada
wanita gestasi multiple atau kehamilan kembar.
Hampir setengah dari sampel penelitian mengalami anemia saat
melahirkan. Prevalensi anemia pada trimester kedua 31,9% dan trimester
ketiga 45,5%. Sedikit data normatif hemoglobin untuk wanita yang
membawa banyak janin. Dalam studi kasus kontrol retrospektif yang cocok
untuknya Paritas. Konsentrasi hemoglobin secara signifikan lebih rendah
selama trimester pertama dan kedua pada anak kembar dibandingkan pada
kehamilan tunggal. Pada sampel penelitian, terjadi perubahan hemoglobin
sepanjang masa gestasi mengikuti relasi kuadrat dengan yang terhebat
Penurunan menjadi jelas saat awal kehamilan dan titik nadir yang terjadi
pada wanita 28 minggu kehamilan. Dimana volume plasma ibu meningkat
50-100% pada wanita dengan gestasi ganda.
Prevalensi anemia kekurangan zat besi yang diamati pada persalinan
adalah 18%, tapi ini hanya menyumbang 30% dari keseluruhan anemia
yang diamati. Kekurangan folat dan vitamin B12 tidak terlihat pada sampel
yang diamati. Namun, pada data populasi ibu hamil dan tidak hamil
Mengaitkan kekurangan vitamin A, selenium, dan vitamin D untuk risiko
anemia. Diperlukan penelitian lebih lanjut untuk mengetahui penyebab
anemia lainnya pada wanita tanpa kekurangan zat besi. Erythropoietin
adalah satu-satunya biomarker besi selama kehamilan yang secara
signifikan terkait dengan hemoglobin saat melahirkan. Erythropoietin
meningkat sebagai respons terhadap anemia, kekurangan zat besi, dan
hipoksia. Dalam penelitian ini, wanita dengan konsentrasi erythropoietin
lebih besar dari persentil ke-75 selama kehamilan sebanyak 3 kali lebih
cenderung menjadi anemia saat melahirkan. Erythropoietin sendiri
menjelaskan 14-28% dari perubahan reseptor transferrin terlarut,
hemoglobin, dan SF dari kehamilan untuk pengiriman ke laboratorium
klinis secara rutin untuk mengukur serum erythropoietin, namun indikator
ini biasanya tidak digunakan untuk mengidentifikasi wanita yang berisiko
kekurangan zat besi atau anemia. penelitian menunjukkan bahwa
eritropoietin merupakan penentu kuat zat besi status dan anemia saat
melahirkan, dan indikator ini tidak terpengaruh dengan peradangan.
Salah satu kekuatan dari penelitian ini adalah bahwa 64% wanita
memiliki longitudinal ukuran status zat besi di masa gestasi dalam hal ini
populasi berisiko rendah. Studi pada penelitian berbeda dari Studi
sebelumnya tentang status besi dalam kelipatan karena penggunaan
biomarker besi ganda, penilaian peradangan, dan tindak lanjut longitudinal
untuk pengiriman. Asumsi pada penelitian ini adalah wanita di Central New
 York daerah yang mendapat perawatan prenatal di Strong Memorial and
Highland Rumah sakit diperkirakan 66% dari mereka yang memiliki
banyak kehamilan serupa dengan 34% wanita yang tersisa, terlihat di
rumah sakit lain di Central New York. Pada penelitian tidak didapatkan
data normatif dari populasi yang cocok untuk tujuan perbandingan, dan
tidak ditemukan data untuk menilai faktor penentu anemia dan kekurangan
zat besi ibu. Data kepatuhan tambahan didasarkan pada laporan sendiri dan
hanya dikumpulkan pada awal. Sampel darah diperoleh selama kunjungan
prenatal yang dijadwalkan untuk tujuan kenyamanan. Dengan demikian,
Waktu penilaian ini tidak distandarisasi dan longitudinal ukuran tidak
tersedia di semua sampel. Sehingga, penentuan volume plasma tidak
diperoleh dan variabilitas dalam ekspansi volume darah akan
mempengaruhi interpretasi biomarker selama kehamilan.
6. Simpulan
Anemia dan zat besi sangat umum terjadi pada wanita dengan
beberapa gestasi. Wanita dengan gestasi multipel menunjukkan prevalensi
anemia dan zat besi yang tinggi. Zat besi ibu dan anemia selama jendela
kritis kehamilan telah terbukti mempengaruhi janin pertumbuhan dan
perkembangan, dan peningkatan data mendukung sebuah asosiasi antara zat
besi ibu dan neonatal yang tidak memadai saat lahir. Karena korelasi yang
sangat signifikan antara status zat besi di awal masa kehamilan dengan zat
besi yang terkuras dan anemia saat persalinan. Maka diperlukan tambahan
skrining dan suplementasi zat besi pada populasi berisiko tinggi sehingga
intervensi dini dan pengobatan suplementasi zat besi dapat diberikan.
Untuk itu, masih diperlukan penelitian tambahan untuk mengidentifikasi
jumlah zat besi tambahan yang dibutuhkan oleh wanita dengan gestasi
multiple yang mengatasi kemungkinan dampak status zat besi ibu dan
neonatal dalam populasi obstetrik berisiko tinggi dan juga menyelidiki
etiologinya.
 C. ARTIKEL JURNAL

Iron deficiency and anemia are prevalent in women with multiple

gestations1 , 2
3 4 4 4 4 3 4
Yuan Ru, Eva K Pressman, Elizabeth M Cooper, Ronnie Guillet, Philip J Katzman, Tera R Kent, Stephen J Bacak,
3
and Kimberly O O’Brien *

3
Division of Nutritional Sciences, Cornell University, Ithaca, NY; and 4School of Medicine, University of Rochester, Rochester, NY
ABSTRACT Keywords: anemia, hepcidin, iron deficiency, multiple
births, pregnancy
Background: Little attention has been placed on the unique iron
demands that may exist in women with multiple gestations. This
merits attention because iron deficiency (ID) during pregnancy is
associated with adverse pregnancy outcomes that are known to be
more prevalent in multiple births.
Objective: We characterized longitudinal changes in iron status
across pregnancy in a cohort of healthy women with multiple INTRODUCTION
gestations and identified determinants of maternal ID and anemia.
In the United States, national survey data have
Design: A group of 83 women carrying twins, triplets, or
quadruplets (aged 20–46 y) was recruited from 2011 to 2014. indicated that anemia and iron deficiency (ID)5 are
Blood samples obtained during pregnancy (w24 wk; n = 73) and at evident in 5.4% and 18% of pregnant women who are
delivery (w35 wk; n = 61) were used to assess hemoglobin, serum carrying singletons, respectively (1). Anemia and ID
ferritin (SF), soluble transferrin receptor (sTfR), hepcidin, serum during pregnancy have been associated with increased
iron, erythropoietin, serum folate, vitamin B-12, C-reactive
protein, and interleukin-6.
risk of a number of adverse birth outcomes including
Results: The prevalence of tissue ID (sTfR .8.5 mg/L) increased
preterm birth (PTB), low birth weight (LBW), and low
significantly from pregnancy to delivery (9.6% compared with 23 neonatal iron stores at birth (2–4). Although the
%, P = 0.03). Women with depleted iron stores (SF ,12 mg/L, n = optimal amount of storage iron at birth is unknown,
20) during pregnancy had a 2-fold greater risk of anemia at growing data suggest that the developing fetal brain is
delivery, and 25% (n = 5) developed iron deficiency anemia (IDA).
susceptible to iron insufficiency in utero, and low iron
Overall, 44.6% of women studied (n = 37/83) were anemic at
delivery, and 18% of women (n = 11/61) had IDA. Erythropoietin stores at birth have been associated with motor-
during pregnancy was significantly negatively associated with cognitive deficits in infants (5–8).
hemoglobin at delivery. Women with erythropoietin .75th Despite the growing body of research on iron
percentile during pregnancy exhibited a 3-fold greater risk of
homeostasis during pregnancy, very little is known
anemia, suggesting that erythropoietin is a sensitive predictor of
anemia at delivery. Inflammation was present at delivery, which about iron status in women with multiple gestations, a
limited the utility of ferritin or hepcidin as iron-status indicators at population that may have higher iron demands during
by guest on November 16, 2017

delivery.
pregnancy. This possibility is of concern because
Conclusions: ID and anemia are highly prevalent in women with
multiple gestations. Additional screening and iron multiple births comprise 3.5% of all births in the
supplementation may be warranted in this high-risk population United States (9), and this group alone is responsible
given the known associations between ID anemia and adverse
for 15% of PTBs and contributes 20% of the LBW
maternal and neonatal outcomes. This trial was registered at
clinicaltrials.gov as NCT01582802. Am J Clin Nutr 2016;104:1052– infants who are born in the United States (10).
60.
Current prenatal recommendations advocate the use
of an ironcontaining prenatal supplement across
gestation, and women with multiple gestations are
advised to adhere to the same guidelines as women
who are carrying singletons. It is unknown ifthe typical
iron content of a standard prenatal supplement (27
mg) is sufficient for all higher-risk obstetric groups. To
date, few data on iron status and anemia exist in
otherwise healthy women with multiple gestations
(11, 12) or in women with multiple gestations who
experienced pregnancy complications (13, 14). The
last published American College of Obstetricians and
Gynecologists practice bulletin on anemia in
pregnancy did not identify women who were carrying
multiple fetuses as an at-risk
1
Supported by the Gerber Foundation.
2 Supplemental Figure 1 and Supplemental Tables 1 and 2 are available
from the “Online Supporting Material” link in the online posting of the
article and from the same link in the online table of contents at
http://ajcn.nutrition.org.
*To whom correspondence should be addressed. E-mail: koo4@cornell.
edu.
5
Abbreviations used: ART, assisted reproductive technology; CRP, C-
reactive protein; GA, gestational age; GWG, gestational weight gain; ID,
iron deficiency; IDA, iron deficiency anemia; IOM, Institute of Medicine;
IVF, in vitro fertilization; LBW, low birth weight; ppBMI, prepregnancy BMI;
PTB, preterm birth; SF, serum ferritin; sTfR, soluble transferrin receptor;
TBI, total body iron.
Received October 29, 2015. Accepted for publication July 29, 2016.
First published online August 31, 2016; doi: 10.3945 /ajcn. 115.126284.
1052 Am J Clin Nutr 2016;104:1052–60. Printed in USA. 2016 American Society for Nutrition
obstetric population and made no additional iron
intake recommendations for this group (15).
To address this current knowledge gap, the goal of this
study was to characterize longitudinal changes in
maternal iron status across pregnancy in a cohort of
healthy women with multiple gestations and to
identify significant determinants of maternal ID and
anemia by exploring relations between iron-status
indicators, hepcidin, and inflammatory indicators
across pregnancy and at delivery.
METHODS
Participants
A cohort of 86 women aged 20–46 y who were
carrying twins (n = 66), triplets (n = 19), or quadruplets
(n = 1) was recruited to participate in a prospective
longitudinal study that was designed to characterize
changes in maternal iron status across gestation. This
trial was registered at clinicaltrials.gov as
NCT01582802.
All participants were recruited between 2011 and
2014 from Strong Memorial Hospital and Highland
Hospital at the University of Rochester. This maternity
program serves as a referral center for women with
multiple gestations in central and upstate New York.
According to the Finger Lakes Regional Perinatal
Downloaded from ajcn.nutrition.org
Database, 66.3% of women who are carrying twins
and all women who are carrying higher-order
multiples receive prenatal care at Strong Memorial
Hospital and Highland Hospital. All healthy women
($19 y) who were carrying multiple fetuses with
uncomplicated pregnancies were approached and
asked to participate in the study to ensure that the
study population was representative of the patient
by guest on November 16, 2017
Supplemental Material can be found at:
http://ajcn.nutrition.org/content/suppl/2016/08/31/ajcn.115.1
26284.DCSupplemental.html
population who were attending the clinic. Women
were excluded if they had HIV infection, preexisting
diabetes mellitus, eating disorders, malabsorption
diseases, hemoglobinopathies, or other medical
problems that were known to affect iron homeostasis
at the time of enrollment. The final population
included 83 women as detailed in Supplemental Figure
1. Informed written consent was obtained from all
participants at baseline. The study was approved by
the institutional review boards of the University of
Rochester and Cornell University.
Demographic and health details were self-reported at
study entry with the use of a health-survey
questionnaire that was constructed for the study. A
prenatal supplement questionnaire that was designed
for the study was also administered at entry into the
study to assess the type and frequency of prenatal
supplement use. Gestational age (GA) was determined
on the basis of a self-reported menstrual history or the
date of in vitro fertilization (IVF). In non-IVF patients, if
the GA that was calculated from the self-reported
menstrual history and that derived from the earliest
ultrasound data differed by .10 d, GA was determined
with the use of the earliest ultrasound estimate.
On the basis of WHO criteria, women were
categorized as underweight, normal weight,
overweight, or obese at entry into pregnancy with the
use of prepregnancy BMI (ppBMI; in kg/m2) (16). A
2009 Institute of Medicine (IOM) committee set
weightgain guidelines for twin pregnancies but only
for women who delivered at term. Because the
majority of our study population delivered
prematurely, the adequacy of gestational weight gain
(GWG) was determined with the use of the IOM
guidelines with adjustment for GA at delivery (17).
Because of the lack of weight-gain guidelines for
underweight women carrying multiple fetuses,
underweight women (n = 4) were categorized with the
use of the same IOM guidelines that were established
for normalweight women. GWGs of women who were
carrying triplets or quadruplets were also categorized
with the use of the same guidelines for twins because
of a lack of GWG criteria for higherorder multiples.
Other maternal data including gestational diabetes
mellitus, pregnancy-induced hypertension, and mode
of delivery were abstracted from medical records. All
collected data were entered and checked by trained
study personnel.
Maternal serum collection and biochemical analyses
Maternal nonfasting blood (15 mL) was obtained for
study purposes during a scheduled prenatal visit or at
delivery. In these research samples, whole blood was
used to assess maternal hemoglobin, and serum was
separated and stored at 2808C for the subsequent
analysis of iron-status indicators, key inflammatory
markers, and serum folate and vitamin B-12. All serum
samples were assayed in duplicate.
Additional longitudinal data on hemoglobin
concentrations across gestation were abstracted from
medical charts because hemoglobin was routinely
assessed as standard of care when blood was drawn
for a-fetoprotein testing at 16–20 wk of gestation and
again when oral glucose tolerance was evaluated at
ajcn.nutrition.org
26–28 wk of gestation. Iron-status indicators were
only measured in blood obtained for research
purposes.
All hemoglobin measures were analyzed with the use
of the Cell Dyn 4000 system (Abbott Laboratories) and
categorized according to the trimester in which the
measurement was obtained.
AnemiawascharacterizedwiththeuseofCDCtrimestersp
ecific definitions as hemoglobin concentrations ,11.0
g/dL in the first and third trimesters and hemoglobin
concentrations ,10.5 g/dL in the second trimester (18).
The severity of anemia was also categorized in all
women with the use of WHO criteria as severe
(hemoglobin concentrations ,7.0 g/dL), moderate
(hemoglobin concentrations from 7.0 to 9.9 g/dL), or
mild anemia (hemoglobin concentrations from 10.0 to
10.9 g/dL) (19). We adjusted the cutoff for anemia
downward for African Americans in our analyses on
the basis of IOM recommendations as 10.2 g/dL for
the first and third trimesters and 9.7 g/dL for the
second trimester (20).
Serum ferritin (SF) and soluble transferrin receptor (
sTfR ) were measured with the use of an ELISA (Ramco
Laboratories Inc.). Depleted iron stores were defined
as an SF concentration ,12.0 mg/L. Tissue ID was
defined when the sTfR concentration was .8.5 mg/L,
which has been shown to have a high specificity for
detecting tissue ID during pregnancy (18–20). Total
body iron (TBI) was calculated with the use of the
published equation
TBIðmg=kgÞ ¼ 2½log10 ðsTfR31000=ferritinÞ
2 2:8229=0:1207 ð1Þ
which has been used in pregnant women (1, 21) with
TBI ,0 mg/kg defined as depleted body iron. Women
with abnormal values for any of the iron-status
indicators (i.e., depleted iron stores, tissue ID, or
depleted body iron) were defined as ID. Iron
deficiency anemia (IDA) was defined as the presence
of ID (on the basis of SF, sTfR, or TBI) and anemia.
Serum hepcidin concentrations were analyzed with
the use of an enzyme immunoassay kit (Bachem).
Values ,0.39 ng/mL were categorized as undetectable,
and in these samples, a value of 0.195 ng/mL was
assigned for analysis purposes. A pooled control
serum sample was run in each assay as an internal
control, and the CV for this sample was 12.2%. Serum
iron was measured with the use of graphite furnace
atomic absorption spectrophotometry (PerkinElmer
AAnalyst 800; PerkinElmer Inc.). A National Bureau of
Standards reference control of bovine liver serum
(standard reference material 1577c) was used as a
control, and values for this standard were #5.0% of
the certified expected value. Serum C-reactive protein
(CRP) concentrations were measured with the use of a
quantikine ELISA kit (R&D Systems). Values .5 mg/L
were defined as high. Serum IL-6 concentrations were
assessed with the use of a high-sensitivity quantikine
ELISA kit (R&D Systems). Erythropoietin, folate, and
vitamin B-12 were analyzed with the use of an
Immulite 2000 immunoassay system (Siemens
Healthcare). Folate insufficiency and vitamin B-12
insufficiency were classified when concentrations
were ,6.8 nmol/L and ,148 pmol/L, respectively (22).
According to current guidelines, as part of their
regular prenatal care, all study participants were
prescribed the same standard prenatal supplement as
recommended for women who are carrying
singletons. Women identified as being anemic were
generally prescribed additional iron supplements (325
mg ferrous sulfate tablets to be taken 2 times/d for a
total of 130 mg supplemental Fe/d). Once additional
iron was prescribed, it was continued over the
remainder of pregnancy because this is the standard
of practice for anemic women in the clinic in
accordance with national recommendations (60–120
mg Fe/d) (20). In anemic women who were prescribed
additional iron supplements, no information was
obtained on the use of or compliance with these
recommendations.
Statistical analysis
The normality of distributions for each continuous
study measure was tested with the use of the Shapiro-
Wilk test. Nonnormally distributed data were
logarithmic transformed, and geometric means were
calculated. Student’s t test and ANOVA were used to
test the normally distributed iron indicators as a
function of maternal characteristics such as age, race,
ethnicity, parity, ppBMI, and the adequacy of GWG.
Wilcoxon’s rank-sum test was used to test for
statistical differences between nonparametric
variables, and the chi-square test of independence
was used for analyses of categorical variables.
Bivariate associations between iron-status indicators
were plotted and assessed with the use of Pearson
correlations. Multivariate regression models were
built to assess the associations between each
ironstatus indicator and hemoglobin concentration
during pregnancy (w24 wk of gestation) and at
delivery. Potential confounders (i.e., maternal race,
GA, and maternal parity) that have been shown to
affect maternal hemoglobin concentration and iron
status were included in the models as covariates. Most
data were analyzed cross-sectionally in the group as a
whole. A longitudinal data analysis was also
performed in the subset of women who provided
multiple blood samples for research measures across
gestation. In all longitudinal analyses, a mixed model
with random coefficients was built to control for the
lack of independence of observations that were due to
repeated measures over time from the same mother.
Logistic regression models were used to identify
potential risk factors for maternal ID. A segmented
regression model was used to determine the cutoff
value of IL-6 at delivery when hepcidin concentrations
started to rise. A sample size of 83 participants was
determined to provide sufficient power (0.80) to
characterize iron status in this population and to
obtain correlations in maternal iron-status indicators
and inflammatory makers of the order of 0.15–0.20
with a = 0.05. Results of statistical tests were
considered significant at P , 0.05. All statistical
analyses were performed with the use of JMP 10.0
software (SAS Institute Inc.).
RESULTS
Maternal characteristics
The characteristics of women enrolled in the study are
presented in Table 1. In women who were using
assisted reproductive technologies (ARTs) (n = 37),
48.6% of women (n = 18) used intrauterine
insemination, 37.8% of women (n = 14) used IVF, and
13.5% of women (n = 5) used clomiphene. In addition,
3 women received egg donations, 5 women received
sperm donations, and one woman received both.
Women who were using ARTs (n = 27) had significantly
higher geometric mean SF [32.4 mg/L (95% CI: 23.0,
45.5 mg/L) compared with 16.4 mg/L (95% CI: 12.9,
20.8 mg/L), respectively; P = 0.001] and lower sTfR
[4.1 mg/L (95% CI: 3.3, 5.1 mg/L) compared with 5.7
mg/L (95% CI: 4.7 , 6.9 mg/L), respectively; P = 0.03]
than their counterparts in the non-ART group (n = 34)
at delivery. Women in the ART group compared with
women in the non-ART group were older at delivery
[mean 6 SD: 33 6 4.4 (n = 37) compared with 28 6 4.5 y
(n = 46), respectively; P , 0.0001], entered prenatal
care earlier in gestation [6.3 6 2.8 (n = 35) compared
with 7.6 6 2.6 wk (n = 43), respectively; P = 0.05], and
all women using ART were married and nonsmokers.
Self-reported data on the frequency of supplement
use was available for 88% of the population (n = 73) at
entry into the study (19.1 6 5.6 wk). Of these women,
82% of subjects (n = 60) stated that they consumed
their supplements daily, 11% of subjects (n = 8)
reported that they consumed their supplements 2–5
times/wk, and 5.4% of women reported that they
either consumed their supplements 1 time/wk (n = 2)
or occasionally (n = 2). Because of the variation in
brands of prenatal supplements that were used,
supplemental iron intake ranged from 27 to 90 mg/d.
Only one woman reported that she did not consume
any prenatal supplements.
On the basis of self-reported ppBMI data, overweight
and obesity were evident in 20% (n = 17) and 33% (n =
27) of the study population, respectively. The
prevalence of obesity in these women before
pregnancy was comparable to the 36.1 % reported in
the 2011–2012 NHANES (23). Pre-eclampsia and
pregnancy-induced hypertension occurred in 22% (n =
18) of these pregnancies. Four women developed
gestational diabetes during pregnancy. The majority of
women who were carrying twins (64%; n = 41)
delivered by cesarean delivery. This prevalence was
slightly lower than the 2008 national prevalence (75%)
in women who were carrying twins (24). In this study,
all but one of the 18 women who were carrying
triplets and the participant who was carrying
quadruplets delivered by cesarean delivery.
Premature delivery was prevalent in this cohort
because nearly 65% of women (n = 54) delivered
before 37 wk of gestation. Of women who delivered
preterm, 16.7% (n = 9) had
 1

Baseline characteristics of women with multiple gestations1

Pregnancy Delivery Pregnancy and Medical chart
blood only2 blood only3 delivery blood4 hemoglobin data
Maternal characteristic Entire cohort only5
n 83 20 8 53 2
Maternal age at entry into prenatal care, y 30.3 6 5.16 30.4 6 5.4 29.9 6 5.9 30.6 6 4.8 22 6 0
Gestational age at entry into prenatal care, wk 19.1 6 5.6 19.4 6 4.5 21.5 6 7.4 18.7 6 5.8 19.5 6 0.7
Race, %
Caucasian 75 85 50 75 50
African American 25 15 50 25 50
Ethnicity, %
Hispanic 6 5 13 6 50
Non-Hispanic 94 95 87 94 50
Parity $1, % 59 55 75 62 50
Prepregnancy BMI, kg/m2 28.2 6 8.1 31.7 6 8,7 24.1 6 5.7 27.8 6 7.8 18.8 6 2.3
Underweight, % 5 0 0 6 50
Normal, % 42 30 75 41.5 50
Overweight, % 20 20 0 24.5 0
Obese, % 33 50 25 28 0
Gestational weight gain, kg 19.6 6 9.2 18.4 6 11.9 21.6 6 8.3 19.3 6 8.4 14.5 6 1.9
Less than recommended, % 33 35 25 30 100
Recommended, % 43 35 50 47 0
More than recommended, % 24 30 25 23 0
Use of ART,7 % 45 50 25 47 0
Gestational age at delivery, wk 34.8 6 2.7 33.2 6 3.4 36.6 6 1.6 35.2 6 2.3 34 6 2.8
Type of multiples, %
Twins 77.1 60 100 79 100
Triplets 21.7 35 0 21 0
Quadruplets 1.2 5 0 0 0
1 No significant differences were evident in baseline characteristics between groups (comparisons were not made for the 2 participants in the medical
chart hemoglobin data–only cohort).
2
Participants who provided serum samples only during pregnancy (24.4 6 5.4 wk).
3
Participants who provided serum samples only at delivery (34.8 6 2.7 wk).
4 Participants who provided serum samples during pregnancy and at
delivery.
5
Participants who only had hemoglobin data abstracted from medical charts.
6
Mean 6 SD (all such values). Data on iron status and inflammatory indicators were
7
ART, assisted reproductive technology.
obtained in research samples that were collected
during pregnancy and at delivery, and these data are
very PTBs (,32 wk of gestation), and one woman had a
presented in Table 2. Overall, 73 women provided
set of extremely preterm triplets (,28 wk of gestation). blood samples during pregnancy, which were
The majority of neonates (71.5%; n = 133) who were
obtained, at 24.4 6 5.4 wk of gestation. In these 73
born to these women had LBW (,2500 g), and 9.7% (n
women, 6 women had research blood drawn during
= 18) were born with very LBW (, 1500 g). The
the first trimester (11.0 6 1.9 wk), 49 women had
neonatal mortality rate in this cohort was 1.6% (3/186 blood drawn during the second trimester (24.2 6 3.0
live births).
wk), and 18 women had blood drawn during the third
trimester (29.5 6 2.2 wk). Delivery blood samples (35.3
Longitudinal changes in iron status across gestation 6 2.3 wk) were collected from 73.5% of women (n =
and at delivery 61).
The prevalence of tissue ID (sTfR concentration .8.5
mg/L) increased significantly from 9.6% (n = 73) during
pregnancy (24.4 6 5.4 wk) to 23% (n = 61) at delivery
(P = 0.03). Of the 73 women who provided blood
samples during pregnancy, 37% (n = 27) of them had
depleted iron stores (SF concentration ,12 mg/L) while
receiving standard prenatal supplementation. The
prevalence of depleted iron stores in our population
was significantly higher than the prevalence reported
for similar trimesters of pregnancy in the NHANES
1999–2006 data during the first [33.3% (n = 2 of 6)
compared with 7.3%, respectively; P = 0.007] and the
second trimester [40.8% (n = 20 of 49) compared with
23.7%, respectively; P = 0.01] (1).
The prevalence of IDA in this population was 15.1% (n
= 73) at 24.4 6 5.4 wk of gestation and did not change
significantly from pregnancy to delivery (18% at 35.3 6
2.3 wk of gestation; n = 61). This lack of a change
between the 2 sampling points might have been due
in part to the fact that premature delivery was
common in these women, the delivery hemoglobin
measures were taken, a mean of 11 wk after the
pregnancy sample, and hemoglobin concentrations
are known to increase slightly in late gestation (25).
Published NHANES data on iron status across
gestation presented data on ID but did not provide
data on IDA, and thus, we could not evaluate our IDA
data in relation to the national NHANES data (1).
All women who were studied had detectable IL-6 and
CRP during pregnancy and at delivery. Although
maternal CRP concentrations did not change
significantly between pregnancy and delivery,
ajcn.nutrition.org
maternal IL-6 concentrations increased significantly
2
Iron-status indicators and hemoglobin concentrations in blood samples obtained for research purposes in women with multiple gestations1

Maternal characteristic Pregnancy2 (24.4 6 5.4 wk)3 Delivery (35.3 6 2.3 wk) P
4
Hemoglobin, g/dL 11.2 6 1.3 (7.1–13.8) [73] 11.2 6 1.5 (7.8–13.6) [61] 0.83
Anemia,5 % (n/total n) 31.5 (23/73) 42.6 (26/61) 0.18
Anemia (adjusted),6 % (n/total n) 27.4 (20/73) 34.4 (21/61) 0.38
sTfR, mg/L 4.1 (3.7, 4.7) [73] 4.9 (4.3, 5.7) (61] 0.06
Tissue ID (sTfR concentration .8.5 mg/L), % (n/total n) 9.6 (7/73) 23.0 (14/61) 0.03
Ferritin, mg/L (n) 15.2 (12.9, 17.9) [73]7 22.2 (17.9, 27.4) (61] 0.005
Depleted iron stores (SF concentration ,12 mg/L), % (n/total n) 37.0 (27/73) 26.2 (16/61) 0.18
TBI, mg/kg 3.2 6 3.4 (25.1–10.4) [73] 3.9 6 4.3 (26.3–12) [61] 0.3
Depleted body iron (TBI ,0 mg/kg), % (n/total n) 12.3 (9/73) 18 (11/61) 0.4
IDA,8 % (n/total n) 15.1 (11/73) 18 (11/61) 0.7
Serum iron, mg/L 13.8 (11.7, 16.4) [72] 15.2 (12.4, 18.4) [56] 0.50
Hepcidin, ng/mL 1.3 (0.9, 1.9) [73] 2.3 (1.7, 3.2) [60] 0.03
Undetectable hepcidin, % (n/total n) 23.3 (17) 3.3 (2) 0.001
Erythropoietin, mIU/mL 30.1 (26.0, 34.9) [72] 34.7 (27.5, 43.9) [59] 0.29
IL-6, pg/mL 1.5 (1.3, 1.7) [71] 5.2 (3.8, 6.9) [57] ,0.0001
.18.4, % (n) 0 (0) 14 (8) ,0.0001
CRP, mg/L 4.5 (3.6, 5.6) [71] 4.4 (3.2, 6.0) [59] 0.85
.5, % (n) 45.1 (2) 42.4 (25) 0.76
Folate, nmol/L NA9 22 (18.8, 25.5) [55] —
,6.8, % (n) NA9 0 (0) —
Vitamin B-12, pmol/L NA9 405.9 (359.2, 458.7) [47] —
,148, % (n) NA9 0 (0) —
1 P values were reported on the basis of statistical differences between pregnancy and delivery (2-tailed t test or chi-square test). CRP, C-reactive
protein; ID, iron deficiency; IDA, iron deficiency anemia; NA, not available; SF, serum ferritin; sTfR, soluble transferrin receptor; TBI, total body iron. 2
Blood samples were obtained for research purposes across all 3 trimesters.
3 Mean 6 SD in parentheses (all such
values).
4 Mean 6 SD; range in parentheses; n in
brackets (all such values).
5
Defined as hemoglobin concentrations ,11.0 g/dL in the first and third trimesters and ,10.5 g/dL in the second trimester.
6
Adjusted for race. Anemia for African American women was defined as hemoglobin concentrations ,10.2 g/dL in the first and third trimesters and ,9.7
g/dL in the second trimester.
7 Geometric mean; 95% CI in parentheses; n in brackets (all such values).
8
Defined as the presence of anemia (adjusted) and ID (tissue ID, depleted iron stores, or depleted body iron).
9
No data were available because the indicator was not analyzed in the pregnancy samples.
across gestation and were significantly associated with only research values were reported. The prevalence of
both SF and hepcidin at delivery only (Supplemental anemia increased significantly from the first to third
Tables 1 and 2). Nearly 10% of the variance in the trimesters (8.1% compared with 45.5%, respectively; P
change in hepcidin from pregnancy to delivery was
, 0.0001). Early anemia was predictive of anemia
explained by IL-6. With the use of a segmented
regression model, we showed that, at delivery, during later gestation as evidenced by the finding that
hepcidin concentrations increased markedly when IL-6 hemoglobin measures that were obtained during
concentrations reached 18.4 pg/mL. either the first trimester (R2 = 0.08, P = 0.01, n = 62) or
second trimester (R2 = 0.38, P , 0.0001, n = 72) were
Longitudinal changes in hemoglobin across gestation significantly positively associated with hemoglobin
and at delivery concentrations at delivery. Women who were anemic
during the second trimester had a nearly 2-fold
Mean hemoglobin concentrations and the prevalence increased risk of anemia (RR: 1.9; 95% CI: 1.1, 3.2) at
of anemia during each trimester are presented in delivery after adjustment for race and the gestational
Table 3. Data in Tables 2 and 3 were obtained from week at delivery. During the second and third
blood samples taken for research purposes. For Table trimesters, 31.9 % of women (n = 72) and 45.5% of
3, when hemoglobin data were available from both women (n = 77) were anemic, respectively. This
the medical charts and the research blood sample,
 3
Hemoglobin concentrations and prevalence of anemia in women with multiple gestations with the use of both blood samples obtained for research
purposes and data abstracted from medical charts1

Trimester 1 (10.3 6 Trimester 2 Trimester 3

1.7 wk)2 (23.5 6 4.0 wk) (30 6 2.5 wk) Delivery (34.8 6 2.7 wk) P
Entire cohort
n 62 72 77 83 —
Hemoglobin, g/dL 12.3 6 1.0 (10–14.2)3,a 10.9 6 1.4 (7.5–13.7)b 11.1 6 1.6 (7.1–14)b 11.2 6 1.6 (7.7–13.9)b ,0.0001
Anemia, % (n/total n) 8.1 (5/62)a 31.9 (23/72)b 45.5 (35/77)b 44.6 (37/83)b ,0.0001
Severe 0 (0/62) 0 (0/72) 0 (0/77) 0 (0/83) —
Moderate 0 (0/62) 18 (13/72) 23.4 (18/77) 21.7 (18/83) 0.54
Mild 8.1 (5/62)a 13.9 (10/72)b 22.1 (17/77)b 22.9 (19/83)b 0.01
IDA,4 % (n/total n) 16.7 (1/6) 14.3 (7/49) 16.7 (3/18) 18 (11/61) 0.64
Caucasian women
n 48 54 57 62
a b b
Hemoglobin, g/dL 12.3 6 1.0 (10–14.2) 11.2 6 1.3 (7.7–13.7) 11.4 6 1.4 (8.6–14) 11.5 6 1.4 (8.8–13.9)b ,0.0001
Anemia, % (n/total n) 10.4 (5/48)a 25.9 (14/54)b 35.1 (20/57)b 32.3 (20/62)b 0.02
IDA, % (n/total n) African 20 (1/5) 17.9 (7/39) 23.1 (3/13) 15.9 (7/44) 0.46
American women
n 14 18 20 21
a b b
Hemoglobin, g/dL 12.2 6 0.7 (11.4–13.7) 10.3 6 1.3 (7.5–12) 10 6 1.5 (7.1–13.4) 10.2 6 1.6 (7.7–13.5)b ,0.0001
Anemia, % (n/total n) 0 (0/14) 50 (9/18) 75 (15/20) 81 (17/21) 0.09
Anemia (adjusted),5 % (n/total n) 0 (0/14) 16.7 (3/18)a 50 (10/20)b 52.4 (11/21)b 0.04
IDA, % (n/total n) 0 (0/1) 0 (0/10) 0 (0/5) 23.5 (4/17) 0.23
1 For any given trimester, when hemoglobin data were available from both the medical charts and from the research blood, only the research value
was reported. Anemia was defined as hemoglobin concentrations ,11.0 g/dL in the first and third trimesters and ,10.5 g/dL in the second trimester. Severe
anemia was defined as a hemoglobin concentration ,7.0 g/dL; moderate anemia was defined as a hemoglobin concentration from 7.0 to 9.9 g/dL; and mild
anemia was defined as a hemoglobin concentration from 10.0 to 10.9 g/dL. IDA was defined as the presence of anemia and iron deficiency (on the basis of
serum ferritin, soluble transferrin receptor, or total body iron). Sample sizes presented are the number of women with IDA of the total number of women
who had both hemoglobin- and iron-status indicator data available within each time period presented. Mean 6 SD gestational weeks of IDA were as
follows: first trimester, 11.0 6 1.9 wk (n = 6), second trimester, 24.2 6 3.0 wk (n = 49), third trimester, 29.5 6 2.2 wk (n = 18), and delivery, 35.3 6 2.3 wk (n =
61).
Different superscript letters indicate significant differences between different trimesters (P , 0.05; ANOVA or chi-square test).
prevalence of anemia was significantly higher than in decline in hemoglobin was fastest during the earlier
the national NHANES data (1999–2006) in which 2.2 % stages of pregnancy (weeks 5–28) and reversed during
(P , 0.0001) and 10.8% (P , 0.0001) of women who late pregnancy (.30 wk) (Figure 1).
were carrying singletons had anemia in the second 2
Mean 6 SD in parentheses (all such values).
and third trimesters, respectively (1). 3
Mean 6 SD; range in parentheses (all such values).
4
IDA, iron deficiency anemia.
5
In the 31.9% of women (n = 23 of 72 studied) who Adjusted for race (20.8 g/dL) for African American women.

were anemic when studied during the second

trimester (23.5 6 4.0 wk), 73.9 % (n = 17) remained
anemic at delivery (34.2 6 3.4 wk). In the 45.5% of
women (n = 35 of 77 studied) who were anemic during
the third trimester (30 6 2.5 wk), 82.9% (n = 29)
remained anemic at delivery (34.7 6 3.3 wk). To
further explore longitudinal changes in maternal
hemoglobin concentrations across pregnancy at the
individual level, a random-intercept and slope model
was applied in women with repeat measures (n = 83).
Maternal hemoglobin concentrations decreased
significantly as pregnancy progressed (b 6 SE = 20.02 6
0.008; P = 0.01). A quadratic relation was observed
between maternal hemoglobin and the gestational
stage of pregnancy, which indicated that the rate of
With the use of the IOM race-adjusted definitions of
anemia (20), the prevalence of anemia in African
Americans was 1.4-fold and 1.6-fold higher than that
observed in Caucasian women during the 3rd
trimester (30 6 2.5 wk) and at delivery (34.8 6 2.7 wk),
respectively.
Determinants of hemoglobin and iron status at
delivery
In women with depleted iron stores (SF concentration
,12 mg/L) during pregnancy (24.4 6 5.4 wk; n = 27),
74% of subjects (n = 20) had both hemoglobin- and
iron-status indicator data (SF, sTfR, or TBI) that were
available at delivery. Of these 20 women, 25% of 35.9 mg/L) (n = 36), respectively; P = 0.03], lower
subjects (n = 5) developed IDA, and 65% of subjects (n hepcidin [1.3 ng/mL (95% CI: 0.9, 2.3 ng/mL) (n = 22)
= 13) were anemic at delivery. Women with depleted compared with 3.6 ng/mL (95% CI: 2.4, 5.4 ng/mL) (n =
iron stores during pregnancy had a 2-fold greater risk 35), respectively; P = 0.004], and higher erythropoietin
of anemia at delivery (RR: 2.0; 95% CI: 1.1, 3.8). [48.4 mIU/mL
Erythropoietin concentrations that were measured
during pregnancy (at w24 wk) were significantly
negatively associated with maternal hemoglobin at
delivery (w35 wk) after adjustment for maternal race,
GA, and maternal parity (R2 = 0.18, P = 0.005, n = 72).
Women who had an erythropoietin concentration
during pregnancy greater than the 75th percentile
(49.7 mIU/mL; n = 16) exhibited 3-fold higher risk of
anemia at delivery (RR: 3.0; 95% CI: 1.7, 5.3).
Erythropoietin was also significantly negatively
associated with SF hepcidin and serum iron and
positively associated with sTfR at delivery
(Supplemental Table 2). Erythropoietin alone
explained 28%, 24%, and 14% of the changes in sTfR,
hemoglobin, and SF, respectively, from pregnancy to
delivery. Unlike ferritin and hepcidin, erythropoietin
concentrations were not confounded by inflammation
because erythropoietin was not associated with either
IL-6 or CRP during pregnancy or at delivery.

Parity was significantly associated with maternal iron

status at delivery. At delivery, women with parity $1 (n
= 34) compared with primagravid subjects (n = 49) had
significantly lower SF [18.1 mg/L (95% CI: 14.0, 23.4
mg/L) compared with 31.7 mg/L (95% CI: 22.6, 44.6
mg/L), respectively; P = 0.01] and hepcidin [1.6 ng/mL
(95% CI: 1.2, 2.4 ng/mL) compared with 4.2 ng/mL
(95% CI: 2.3, 7.6 ng/mL), respectively; P = 0.007] and
significantly higher sTfR [5.9 mg/L (95% CI: 5.0, 6.9
mg/L) compared with 3.6 mg/L (95% CI: 2.8, 4.7 mg/L),
respectively; P = 0.002]. In addition, marital status was
significantly associated with hemoglobin status and
iron status at delivery. Single women compared with
their married counterparts at delivery had significantly
lower hemoglobin concentrations [10.6 6 1.6 (n = 30)
compared with 11.6 6 1.5 g/dL (n = 50), respectively; P
= 0.006] , lower SF [16.8 mg/L (95% CI: 12.2, 23.2
mg/L) (n = 22) compared with 27.3 mg/L (95% CI: 20.7,
 IRON DEFICIENCY AND ANEMIA IN MULTIPLE BIRTHS 1069
TABLE

FIGURE 1 Longitudinal changes in hemoglobin concentrations across pregnancy in 83 women who were carrying multiple fetuses (dashed black line
with SDs) were compared with CDC reference mean values (solid black line). The dotted line represents the fifth percentile of the CDC reference data. A
quadratic relation between hemoglobin and the gestational week was shown in these women who were carrying multiple fetuses with the use of a
nonlinear regression model. Maternal hemoglobin concentrations decreased significantly as pregnancy progressed (b6 SE = 20.02 6 0.008; P = 0.01) with
the rate of decline being steepest during the earlier stage of pregnancy (weeks 5–25 of gestation) and reversing slightly during late pregnancy (.30 wk of
gestation). The figure is based on the original data obtained from reference 27 and adapted from reference 25 with permission.

(95% CI: 35.2, 66.5 mIU/mL) (n = 21) compared with 27.4 mIU/mL (95% CI: 20.1, 37.4 mIU/mL) (n = 35),
respectively; P = 0.02]. Hemoglobin, sTfR, and SF concentrations at delivery were not significantly associated
with maternal age at delivery, ethnicity, or self-reported prenatal supplement use.

DISCUSSION

To the best of our knowledge, this is the largest study to explore maternal ID and anemia in women who were
carrying multiple fetuses. Both ID and anemia were prevalent in this high-risk maternity population.
Anemiawas shownin45% ofwomen during the third trimester, which was 4-fold higher than the expected
national prevalence reported for women who were carrying singletons (1). Although early ID was a significant
determinant of anemia at delivery in women with multiple gestations, there are no early screening guidelines
for this group, and they are not in the high-risk groups that have been identified by the last published
American College of Obstetricians and Gynecologists Practice Bulletin onanemiainpregnancy (15).
Erythropoietin was shownto be the strongest determinant of maternal hemoglobin at delivery. Because of the
high correlations shown between erythropoietin during pregnancy and hemoglobin at delivery and the
findings that erythropoietin was not affected by maternal inflammation, screening for elevated erythropoietin
during pregnancy may help to identify women who are at higher risk of developing anemia at delivery.

Maternal ID was prevalent in women with multiple gestations with tissue ID increasing significantly from
pregnancy to delivery. A significantly higher prevalence of depleted iron stores was shown during the first and
second trimesters than in 1999–2006 NHANES data (1). Of women with depleted iron stores during pregnancy,
more than one-half were shown to be anemic w10 wk later when they delivered their infants. Note that,
similar to national data (9), 65% (n = 54) of our population delivered prematurely, and the high prevalence of
anemia and ID noted at delivery were shown roughly 5 wk earlier than for a typical term gestation (40 wk).
Anemia is known to increase risks of PTB and LBW (2), which are adverse birth outcomes that are highly
prevalent in women who are carrying multiple fetuses. The degree to which maternal anemia exacerbates risks
of PTB and LBW in this group is unknown and merits further study.

Nearly one-half of this study population was anemic at delivery. The prevalences of anemia during the second
trimester (31.9%; n = 23) and third trimester (45.5%; n = 35) were significantly higher than in the national
NHANES data (1999–2006) in women who were carrying singletons (1). Few normative hemoglobin data exist
for women who are carrying multiple fetuses. In a retrospective case-control study that was matched for
parity, significantly lower hemoglobin concentrations were evident during the first and second trimesters in
twins than in singleton pregnancies (11). In our population, changes in hemoglobin across gestation followed a
quadratic relation with the greatest decline being evident during early gestation and a nadir that occurs at w28
wk of gestation at which time maternal plasma volume has been reported to be increased by 50–100% in
women with multiple gestations (26). Compared with the CDC reference curve on the basis of iron-
supplemented singleton pregnancies (25, 27), the hemoglobin curve in our study population was shifted
downward at all GAs with a markedly steeper decrease in early gestation.

The observed prevalence of IDA at delivery was 18%, but this value accounted for only 30% of the overall
anemia observed. Folate and vitamin B-12 deficiencies were not evident in this population.
Growingdatainpregnantand nonpregnantpopulations are linking deficiencies of vitamin A (28), selenium (29),
and vitamin D (30) to risk of anemia. Further studies are needed to explore other causes of anemia in women
without ID. Normative data on IDA inpregnant women were not reported in the NHANES (1) or CDC (31)
studies; thus, we were unable to compare our IDA prevalence to that in national data.

Erythropoietin was the only iron biomarker during pregnancy that was significantly associated with
hemoglobin at delivery. Erythropoietin increases in response to anemia, ID, and hypoxia (32). In our study,
women with erythropoietin concentrations greater than the 75th percentile during pregnancy were 3 times
more likelytobeanemicatdelivery.Erythropoietinaloneexplained 14–28% of the change in sTfR, hemoglobin,
and SF from pregnancy to delivery. Clinical laboratories routinely measure serum erythropoietin, but this
indicator is not typically used to help identify women who are at subsequent risk of ID or anemia. Our study
showed that erythropoietin was a strong determinant of iron status and anemia at delivery, and this indicator
was not affected by inflammation.

Although SF is commonly used to diagnose ID, there are limitations to using SF under inflammatory conditions
(33, 34). In our study population, IL-6 was positively associated with both SF and hepcidin at delivery, which
was consistent with earlier findings in teen pregnancies (35). Serum IL-6 upregulates hepcidin synthesis and
this cytokine is frequently used when examining associations between inflammation and hepcidin. In our
study, hepcidinconcentrations were significantly higherinsubjects with IL-6 concentrations .18.4 pg/mL at
delivery. Because 92% of the study population exhibited increases in IL-6 from pregnancy to delivery, these
findings highlight the need to include a marker of inflammation when interpreting iron-status indicators that
also function as acute-phase proteins (ferritin and hepcidin) to avoid underestimating the prevalence of ID.

One strength of this study was that 64% of women had longitudinal measures of iron status across gestation in
this understudied high-risk population. Our study is distinct from the previous studies on iron status in
multiples (12, 36) because of our use of multiple iron biomarkers, the assessment of inflammation, and the
longitudinal follow-up to delivery. Some study limitations should be noted. We assume that women in the
Central New York area who received prenatal care at Strong Memorial and Highland Hospitals (estimated to be
66% of those carrying multiple gestations) were similar to the remaining 34% of women who were seen at
other hospitals in Central New York. We did not obtain normative data from a matched population who were
carrying singletons for comparison purposes, and we did not obtain dietary data to assess dietary
determinants of maternal anemia and ID. Supplement-compliance data were based on self-report and
collected only at baseline. Blood samples were obtained during scheduled prenatal visits for convenience
purposes, and thus, the timing of these assessments was not standardized, and longitudinal measures were
not available in all study participants. Finally, plasma volume determinations were not obtained, and the
variability in blood volume expansion would have affected the interpretation of biomarkers during pregnancy.
A U-shaped distribution between hemoglobin at term and increased risk of adverse birth outcomes have been
shown, which may in part have been due to an altered plasma volume expansion (37–40).

In conclusion, women with multiple gestations exhibit a high prevalence of anemia and ID. Maternal ID and
anemia during critical windows of pregnancy have been shown to affect fetal growth and development, and
increasing data support an association between maternal ID and insufficient neonatal iron stores at birth (41,
42). Because of the highly significant correlations between iron status early in gestation with depleted body
iron and anemia at delivery, early screening for maternal iron status is warranted in this high-risk population
so that early intervention and iron-supplementation regimens can be initiated when warranted. Additional
studies are needed to identify the amount of supplemental iron that is required by women carrying multiple
fetuses to address the possible impact of maternal iron status on neonatal iron stores in this high-risk obstetric
population and to investigate the etiology of non-IDA.

The authors’ responsibilities were as follows—YR: analyzed the data; YR, EKP, EMC, RG, TRK, and KOO: designed and conducted the research; YR and
KOO: wrote the manuscript; KOO: had primary responsibility for the final content of the manuscript; and all authors: revised the manuscript and read
and approved the final manuscript. The Gerber Foundation had no conflict of interest in the study and was not involved in the study design or
interpretation of the data. None of the authors reported a conflict of interest related to the study.

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D. OUTLINE TINJAUAN PUSTAKA
1. Judul Penelitian : Hubungan Defisiensi Zat Besi dan Anemia Terhadap
Ibu Hamil Dengan Gestasi Multiple
2. Outline Tinjaun Pustaka
a. Ibu Hamil Dengan Gestasi Multiple
1) Karakteristik Ibu
a) Obesitas
b) Usia
c) Ras
d) Suku
e) Paritas
2) Prevalensi IDA yang diamati pada saat bersalin
3) Perubahan Longitudinal Pada Hemoglobin Selama Kehamilan Dan Saat
Melahirkan
b. Defesiensi Zat Besi dan Anemia
1) frekuensi penggunaan suplemen
2) Faktor penentu iron deficiency ibu hamil dan anemia
3) kekurangan vitamin A, selenium, dan vitamin D
c. Hubungan Defesiensi Zat Besi dan Anemia terhadap Ibu Hamil Dengan
Gestasi Multiple

E. KERANGKA KONSEP

Variabel Dependen Variabel Independen

Defesiensi Zat Besi Ibu Hamil Dengan
dan Anemia Gestasi Multiple

Variabel Pengganggu
1. Karakteristik Ibu
2. Faktor penentu iron
deficiency ibu hamil dan
anemia
3. Perubahan Longitudinal
Pada Hemoglobin
Selama Kehamilan Dan
Saat Melahirkan
 Keterangan :

: Variabel yang diteliti

: Variabel yang tidak diteliti

: Arah penelitian

F. DEFENISI OPERASIONAL
Definisi operasioanal adalah uraian tentang batasan variabel secara
operasional berdasarkan karakteristik yang diamati (Notoadmojo,2012).

Variabel Defenisi Operasional Alat Ukur Kriteria Objektif Skala

1. Defesiensi Asupan zat besi Kuesioner Baik : 76-100% Ordinal
Besi dengan jumlah yang Cukup : 56-75%
cukup sebesar 95%. Kurang : <56%

2. Anemia Kondisi ibu dengan Kuesioner Anemia : <11 Nominal

kadar Hemoglobin gr%
kurang dari 11 gr% Tidak Anemia :
>11 gr%

3. Ibu Hamil
Kekurangan zat besi Kuesioner Beresiko : <50% Nominal
dengan
pada ibu dengan Tidak Beresiko :
Gestasi
kehamilan multipel >50%
Multipel
meningkat signifikan
sampai persalinan.