Adverse maternofoetal outcomes associated with ionised calcaemia, total calcaemia, albuminaemia, and calcium supplementation in pregnancy: Analysis from a resource-limited setting.

INTRODUCTION: Disorders of total calcium (tCa) in pregnancy have been associated with adverse maternofoetal outcomes. However, studies evaluating this from the viewpoint of ionised calcaemia are practically inexistent. This study estimates the prevalence of some adverse maternal and foetal outcomes and the potential effect of ionised calcium (iCa), tCa, albumin and calcium supplementation on some maternofoetal outcomes.
METHODS: A cross-sectional study was conducted among 1074 pregnant women in late pregnancy from four health facilities in the Nkongsamba Health District. Data were collected by interview, analysis of maternal blood samples and measurement of maternal and foetal parameters. Total calcaemia and albuminaemia were measured by atomic absorption spectrophotometry, while iCa and pH were measured using ion-selective potentiometry. Associations were measured using the odds ratio in simple and multiple logistic regression.
RESULTS: The prevalence of low birth weight, macrosomia, and hypertension in pregnancy was 6.27 [4.97-7.89]%, 4.78 [3.65-7.89]%, 10.24 [8.57-12.20]%, respectively. Following multiple logistic regression, women with iCa levels ≤ 1.31mmol/L had significantly increased odds of hypertension in pregnancy (AOR = 2.47 [1.63-3.74], p-value = 0.000), having babies with low birth weight (AOR = 2.02[1.33-3.61], p-value = 0.002), low birth length (AOR = 2.00 [1.34-2.99], p-value = 0.001), low brachial circumference (AOR = 1.41[1.10-1.81], p-value = 0.007), first minute Apgar score < 7 (AOR = 3.08[1.70-5.59], p-value = 0.000) and fifth minute Apgar score < 7 (AOR = 2.86[1.32-6.16], p-value = 0.007). Ionised calcaemia had no significant association with maternal body mass index immediately after birth and the head circumference of the baby. Total calcaemia was found to have no significant association with any of the selected outcomes, while women with total albuminaemia ≤ 30mg/L had significantly higher odds of having babies with low birth weight (AOR = 3.40[1.96-5.91], p-value = 0.000), and Apgar scores < 7 at the first (AOR = 2.07[1.16-3.70], p-value = 0.013). Calcium supplementation showed no significant association with any of the selected outcomes except for the first (OR = 0.42[0.24-0.72], p-value = 0.002) and fifth minute Apgar score (OR = 0.25[0.12-0.50], p-value = 0.000).
CONCLUSION: The prevalence of low birth weight, macrosomia, and hypertension in pregnancy was 6.27 [4.97-7.89]%, 4.78 [3.65-7.89]%, 10.24 [8.57-12.20]%, respectively. Maternal iCa levels ≤ 1.31mmol/L significantly increase the odds of having babies with low birth weight, low birth length, low brachial circumference at birth, low Apgar scores at the first and fifth minutes and maternal hypertension in pregnancy. Low maternal albuminaemia is significantly associated with low birth weight, and Apgar score < 7 at the first minute. None f the selected maternofoetal outcomes directly depend on total calcaemia, given that none of the associations was statistically significant. Even though iCa levels remain relatively normal in normal pregnancies, it remains the strongest predictor of foetal outcomes. Calcium supplementation significantly improves the Apgar scores at the first and fifth minute. Routine pregnancy follow-up should include evaluating maternal calcaemic states, particularly the ionised fraction, to detect the low-normal concentrations likely to impact maternal and foetal outcomes. Normal iCa levels for pregnant women need revisiting.

Introduction

Calcium in the human body contributes to about 1–2% of body weight, with 99% stored in the bone and teeth as hydroxyapatite [1-3]. The remaining 1% (5-6g) is found in the intracellular and extracellular milieu, with only 1.3g found in the extracellular milieu. In plasma (part of the extracellular milieu), calcium circulates in three principal forms. About 50% is free ionised calcium (iCa) and constitutes the metabolically active form of calcium, while 40% is bound and transported by plasma proteins (principally albumin). The remaining 10% is bound to small anions such as phosphate, carbonate, citrate, lactate, and sulphate [4, 5].

Hypocalcaemia in pregnancy is indisputably a widespread finding in pregnancy, particularly from the perspective of total calcaemia. In low and middle-income countries, the prevalence is very high, especially in the third trimester, where foetal calcium demands are highest [6]. The third-trimester prevalence varies from 58% in Cameroon [7], 70% in Algeria [8] and 66% in India [9]. According to literature, even though total calcium (tCa) levels vary and go below the normal range in pregnancy, iCa levels are usually maintained within the normal range [6]. The measurement of iCa levels is complicated and liable to be influence by multiple factors. Moreover, the belief that iCa levels remain normal in pregnancy has discouraged research using ionised calcaemia in pregnancy and its routine use in clinical practice.

Low calcium intake significantly increases the likelihood of hypertensive diseases in pregnancy (gestational hypertension, pre-eclampsia and eclampsia) [10, 11], leads to low birth weight (LBW), low birth length (LBL) and small for gestational age babies [12]. In a recent study in India, total hypocalcaemia was significantly associated with LBW but had no statistically significant association with preterm delivery, pre-eclampsia and neonatal mortality [13]. Similar findings showing a non-statistically significant association between pre-eclampsia and total hypocalcaemia have been reported in other studies [14, 15]. The above findings remain controversial as contrasting evidence suggests a significant association between calcium levels and hypertensive diseases in pregnancy [16-18].

Literature evaluating the association between calcium supplementation in pregnancy and maternal and foetal outcomes like hypertensive diseases in pregnancy, and foetal birth weight (FBW) is available. However, studies that explore the relationship between total blood calcium levels (talk less of ionised calcaemia), and these outcomes are still sparse. In a very recent study where iCa was measured, ionised calcaemia was found to have a more robust prediction for pre-eclampsia than total calcaemia [19]. Most of these studies have been carried out around hypertensive diseases in pregnancy, and only a few have tried to verify which calcium fraction best predicts which outcomes. Moreover, studies comparing the effect of total calcaemia and ionised calcaemia on other foetal outcomes like BW, Apgar score (AS), brachial circumference (BC), and head circumference (HC) are practically inexistent.

Therefore, this write-up determines the potential effect of maternal blood calcium levels (tCa and iCa levels) and albumin on selected maternofoetal outcomes. It also presents the prevalence of some adverse obstetric outcomes and reassesses outcomes associated with calcium supplementation in pregnancy. This covers objective 3 of the registered research protocol previously published in PLoS one [20]. The manuscript evaluates maternofoetal hypocalcaemia-related outcomes, but given the strong relationship between albuminaemia and calcaemic states, as well as the link between calcium supplementation, calcaemic states and maternofoetal outcomes, the objectives of this manuscript were formulated as stated above. In this way, we will find out if each of the chosen factors had an association with the occurrence of adverse maternofoetal outcomes and also detect factors with a stronger association.

Methodology

The methodology used in this research had been described and published in PLOS One before the onset of this study [20]. The specificities related to this write up have been described here.

Study design and setting

This was a cross-sectional hospital-based study targeting apparently healthy pregnant women in late pregnancy (37 weeks of pregnancy and above) of the Nkongsamba Health District (NHD) between November 2020 and September 2021. The NHD is an extensive health district in the Moungo division, Littoral Region, Cameroon. Contrary to information presented in the original research protocol, four major health facilities of the NHD were considered for data collection in this study instead of the Nkongsamba Regional Hospital (NRH) alone [20]. These health facilities included the NRH, Bon Samaritain Medicalised Health Centre, Catholic Medicalised Health Centre, and the Fultang polyclinic. About 8 in 10 deliveries in this health district occur in these health facilities. These changes were made to have a more representative sample of the NHD. Data were obtained by administering face-to-face, an interviewer-administered semi-structured questionnaire and additional data obtained through maternal biochemical blood assays.

Eligibility, sample size, and sampling

Eligible participants were pregnant women at a gestational age of 37 weeks, and above, who were apparently healthy and were received at the maternities of the four included health facilities for antenatal care. Contrary to reports in the original protocol, which indicated that women with potential causes of hypocalcaemia were to be excluded, all apparently healthy pregnant women were included to have a general picture of the calcium profile in pregnant women of the health district. As presented in the protocol, we conducted this study with a calculated minimum sample size of 1067 participants, and sampling was exhaustive of all eligible and consenting pregnant women received during the study [20].

Procedure of implementation, data collection and biochemical assays

As described in the protocol, after obtaining the required administrative authorisations, the data collection tools were pretested, and ethical clearance was obtained for the study. During three training sessions (5 hours each), seven midwives working in the selected maternities were trained on the study objectives, the consenting and the data collection procedure [20].

Data were collected on a one-to-one basis using a semi-structured interviewer-administered questionnaire. Following the World Health Organisation (WHO) best practice for phlebotomy [21], 10 ml each of blood was drawn into lithium heparinised and dry vacutainer tubes for the measurement of total calcaemia and ionised calcaemia, respectively. This blood collection was done on well-rested participants (10 minutes), seated with no fist formation and no prior physical exercise [22]. The samples were collected using vacuated needles with very short-lived use of tourniquets (less than 1 minute) [20].

Blood in heparinised tubes was used to measure tCa and albumin, while the blood collected in dry vacutainer tubes was settled (anaerobically) for 30 minutes and the serum extracted to measure iCa levels and pH. Total calcaemia and albuminaemia were measured using a semi-automatic KENZA MAX spectrophotometer (by atomic absorption spectrophotometry) following BIOLABO standard operating procedures. Total calcaemia was measured using O-Cresol Phtalein Complexone (CPC) [23], while total albumin measurement used Bromocresol green (BCG) [24] as reagents. Ionised calcaemia and pH were measured by ion-selective potentiometry using the K-lite 8 serum electrolyte analyser (MSLEA15-H model), with the manufacturer’s standard operating procedures and reagents [20]. Measured iCa levels were then corrected for mild pH changes using the formula: Corrected iCa2+ (pH 7.4) = Measured iCa2+ [1–0.53 × (7.40–measured pH)] [25] which is valid between 7.2–7.6. Payne’s equation was used to adjust tCa concentrations for albumin changes; Caadjusted(mmol/L) = tCa (mmol/L) + 0.02 [40 –albumin (g/L)] [26].

The blood pressure (BP) was taken in a seating position after 10 minutes of rest on the day of blood sample collection. Adapted cuffs and aneroid sphygmomanometers were used, with two BP measurements taken (separated by 2 minutes intervals) and the average BP calculated. The mean BP from the arm with the higher average was considered [27].

Second contact was made with participants at delivery for more data collection from the mother-baby pair. The mother’s height and weight, foetal birthweight (FBW), birth length (BL), BC, HC, AS at the first and fifth minute were measured and registered in the questionnaires. Each participant’s height was measured in an erect position, using a graduated height measuring scale (in meters). The weight and height of participants were measured just after birth, with shoes off, light clothing, and empty pockets. Body Mass Index (BMI) was calculated as measured weight minus 1 kg (adjusting for clothing), divided by height squared (kg/m2) [20]. The FBW was measured using a digital baby weighing scale and recorded to the nearest gramme in the questionnaire. The HC, BC, and BL were measured using an adapted measuring tape and recorded to the nearest centimetre in the questionnaire. The AS was evaluated using the five parameters of the APGAR score at the first and fifth minute following delivery.

Data analysis

All questionnaires lacking essential information like ionised calcaemia, total calcaemia, and albuminemia were rejected. Data from retained questionnaires were entered into a predesigned data entry sheet on Epi-info, cleaned and analysed. Proportions and 95% confidence intervals (CIs) were estimated for categorical variables, while the means and standard deviations were used for continuous variables. Maternal and foetal variables (outcomes) were transformed into binary variables. LBW was defined as FBW less than 2500g [28], LBL as BL ≤ 48cm [29], low LBC as BC ≤ 11cm, and low AS as AS < 7 [30]. Hypertension in pregnancy was defined as Systolic BP ≥ 140mmHg and or diastolic BP ≥ 90mmHg [27]. To evaluate the prevalence of hypertensive diseases in pregnancy among hypocalcaemic women, a cut-off of 45mg/L and 85mg/L were used for ionised and total hypocalcaemia, respectively. Concerning the factors, ionised calcaemia was made binary using a cut-off of 1.31mmol/L (taken from the mean ionised calcaemia in the study), tCa was categorised using the cut-off of 85mg/L [20], and albuminaemia was categorised using the cut-off of 30mg/L (the mean concentration recorded in the study).

The association between the different factors (like iCa, tCa, albumin and calcium supplementation) and the selected outcomes (like FBW, BL, AS, and maternal hypertension) was measured using the odds ratio (OR) and its 95% CI. Factors with p-values <0.25, were included in the multiple logistic regression model with a statistical significance set at a p-value threshold of 0.05. The OR, Adjusted OR (AOR) and p-value were reported for each outcome.

Ethics statement

Ethical clearance was obtained from the Cameroon Bioethics Initiative/ Ethics Review and Consultancy Committee CAMBIN/ ERCC in Yaounde, with CBI/452/ ERCC/CAMBIN as the reference number. In addition, an information notice was prepared and was used to enlighten each participant on the risk and benefit of the study and what is expected of them. Consent was written and signed before enrollment. Young pregnant girls below 21years of age signed assent forms, and written consent was obtained from their parents, legal representatives or guardians. Participation was at will, and participants were free to leave the study without any penalisation. Only consenting and eligible participants were recruited for this study. All questionnaires were coded and remained confidential.

Results

Characteristics of the study population

A total of 1150 pregnant women in the late third trimester were contacted for this study. However, 76 eligible women refused to participate in the study, giving us a non-response rate of 6.61%. The ages of the participants ranged from 15 to 47 years, with a mean age of 28.20±6.08 years. The sampled population was dominated by pregnant women aged 21–30 years (56.52%), and about 9 in 10 (93.29%) had attended at least secondary school. A majority of these women were cohabiting (39.89%), while 25.19% were legally married (65.09% in a union), and 34.74% single.

The mean total number of pregnancies was 3.46±2.06, while the average gestational age at the first antenatal visit(for the current pregnancy) was 17.10±8.08 weeks. About 2 in 10 women were in their first pregnancy, while most were in their second, third or fourth pregnancy (47.06%). About half (53.52%) initiated antenatal care after 4–6 months of pregnancy, while only about 1 in 10 women (11.62%) started antenatal care within the first two months of pregnancy. Concerning the number of antenatal visits, 87.14% had attended at least 4 antenatal care sessions.

Prevalence of some adverse obstetric outcomes

Table 1 presents maternal and foetal outcomes. According to the results, the prevalence of hypertension in pregnancy was 10.24 [8.57–12.20]%. The prevalence of hypertension in pregnancy among women with hypocalcaemia was also evaluated (not on the table). This prevalence was 11.33 [9.13–13.97]% among women with total hypocalcaemia and 8.50 [6.17–11.59]% among women with total normocalcaemia (p-value = 0.136). As far as ionised hypocalcaemia is concerned, the prevalence of hypertension in pregnancy among women with ionised hypocalcaemia was 23.53 [10.75–41.17]% and 9.81 [8.15–11.77]% among women with ionised normocalcaemia (p-value = 0.009).

Table 1

Prevalence of some adverse maternal and foetal outcomes.

Variable	Modalities	Proportion	Percentage
Hypertension in pregnancy (Systolic blood pressure≥140mmHg and/or Diastolic blood pressure ≥ 90mmHg) (n = 1074)	Yes	110	10.24
	No	964	89.76
Foetal birth weight (n = 1068)	<2500g	67	06.27
	2500g≤BW<3500g	696	65.17
	3500g≤BW<4000g	254	23.78
	≥4000g	51	04.78
Apgar Score at first minute (n = 1068)	< 7	49	04.59
	8–10	1019	95.41
Apgar Score at the fifth minute (n = 1070)	< 7	28	02.62
	8–10	1042	97.38

Concerning the FBW, 6.27 [4.97–7.89]% gave birth to babies with LBW, 23.78% had big babies at birth, while 4.78 [3.65–7.89]% had macrosomic babies (>4000g). Only 2.62 [1.82–3.76] % had babies with a fifth-minute AS < 7.

Association of ionised calcaemia, total calcaemia and albuminaemia with selected maternofoetal outcomes

Table 2 shows the potential associations of calcaemic levels with some maternal and foetal outcomes. As far as LBW is concerned, ionised calcaemia and total albuminaemia havestatistically significant associations with LBW. Following multiple logistic regression, women with iCa levels ≤ 1.31 mmols/L had their odds of having babies with LBW increased by about 2 folds (AOR = 2.19[1.33–3.61], p-value = 0.002) while women with albumin levels ≤ 30 mg/L had their odds to deliver babies with LBW increased by up to 3.40 folds (AOR = 3.40[1.96–5.91], p-value = 0.000). Total calcaemia did not show any statistically significant association with LBW.

Table 2

Association of ionised, total calcaemia and albuminaemia with maternofoetal outcomes.

Outcome	Factors in blood	Bivariate analysis		Multivariate analysis
		OR [CI]	p-value	AOR [CI]	p-value
Low birth weight	Ionised calcium	2.02 [1.25–3.28]	0.004*	2.19[1.33–3.61]	0.002*
	Total Calcium	1.47 [0.88–2.48]	0.137	1.19[0.70–2.03]	0.512
	Albumin	3.21[1.86–5.55]	0.000*	3.40[1.96–5.91]	0.000*
	Albumin-corrected Calcium	1.33[0.81–2.17]	0.261
First-minute Apgar score < 7	Ionised calcium	3.00[1.67–5.39]	0.000*	3.08[1.70–5.59]	0.000*
	Total calcium	1.55[0.85–2.81]	0.150	0.86[0.23–3.23]	0.824
	Albumin	1.91[1.08–3.38]	0.026*	2.07[1.16–3.70]	0.013*
	Albumin-corrected calcium	1.61[0.90–2.86]	0.108	1.49[0.42–5.37]	0.539
Fifth-minute Apgar score < 7	Ionised calcium	2.88[1.35–6.15]	0.006*	2.86[1.32–6.16]	0.007*
	Total Calcium	1.90[0.85–4.27]	0.120	1.64[0.36–7.44]	0.523
	Albumin	1.65[0.80–3.42]	0.175	1.73[0.83–3.61]	0.144
	Albumin-corrected calcium	1.70 [0.78–3.63]	0.168	0.95[0.23–3.91]	0.943
Hypertension in pregnancy	Ionised calcium	2.55[1.68–3.84]	0.000*	2.47[1.63–3.74]	0.000*
	Total Calcium	1.38[0.90–2.10]	0.138	0.88[0.34–2.30]	0.797
	Albumin	0.86[0.58–1.28]	0.467
	Albumin-corrected calcium	1.44[0.96–2.17]	0.081	1.43[0.56–3.63]	0.455
Low birth length (≤48cm)	Ionised calcium	2.04[1.37–3.04]	0.001*	2.00[1.34–2.99]	0.001*
	Total calcium	1.29[0.86–1.96]	0.222	1.18[0.78–1.80]	0.441
	Albumin	1.19[0.80–1.76]	0.383
	Albumin-corrected calcium	1.11[0.74–1.65]	0.616
Brachial circumference ≤ 11cm	Ionised calcium	1.43[1.11–1.84]	0.005*	1.41[1.10–1.81]	0.007*
	Total calcium	0.95[0.73–1.22]	0.679
	Albumin	0.77[0.60–0.99]	0.040*	0.79[0.61–1.02]	0.066
	Albumin-corrected calcium	1.03[0.80–1.33]	0.799
Head circumderence at birth ≤ 35cm	Ionised calcium	0.80[0.63–1.03]	0.080	0.82[0.64–1.05]	0.111
	Total calcium	0.94[0.74–1.22]	0.678
	Albumin	1.29[1.01–1.65]	0.041*	1.27[0.99–1.62]	0.056
	Albumin-corrected calcium	0.99[0.77–1.26]	0.923
Maternal Body mass index after birth above 25kg/m2	Ionised calcium	1.02[0.79–1.33]	0.869
	Total calcium	0.99[0.76–1.29]	0.936
	Albumin	1.03[0.80–1.34]	0.807
	Albumin-corrected calcium	0.97[0.74–1.26]	0.802

*Statistically significant, OR = Odds Ratio, AOR = Adjusted OR, CI = Confidence Interval

Concerning the first minute AS, ionised calcaemia and albuminaemia were found to have a statistically significant association with the AS at the first minute. Following multiple logistic regression, women who had iCa levels ≤ 1.31 mmol/L had their odds of having babies with first-minute AS < 7 increased by 3.08 folds (AOR = 3.08[1.70–5.59], p-value = 0.000), while women with albumin levels ≤ 30mg/L had their odds of having babies with the first-minute AS < 7 increased by 2.07 folds (AOR = 2.07[1.16–3.70], p-value = 0.013). Concerning the fifth-minute AS, only ionised calcaemia had an effect. Following multiple logistic regression, women with iCa levels ≤1.31 mmol/L had their odds of having babies with a fifth-minute AS < 7 increased by 2.86 folds (AOR = 2.86[1.32–6.16], p-value = 0.007). Total calcaemia had no statistically significant association with AS.

Concerning hypertension in pregnancy, only ionised calcaemia had a statistically significant association with hypertension in pregnancy. Following multiple logistic regression, women with iCa levels ≤ 1.31mmol/L had their odds of hypertension in pregnancy increased by 2.47 folds (AOR = 2.47[1.63–3.74], p-value = 0.000). Total calcaemia and albuminaemia did not significantly affect the odds of hypertension in pregnancy.

Concerning the BL, only ionised calcaemia it was found to have an effect. Following multiple logistic regression, women with iCa levels ≤ 1.31mmol/L had their odds of LBL in the babies increased by 2 folds (AOR = 2.00[1.34–2.99], p-value = 0.001). Total calcaemia and albuminaemia did not significantly affect BL. Concerning the BC, only ionised calcaemia was found to have an impact. Women with iCa levels ≤ 1.31mmol/L had their odds of LBC (≤11cm) in the baby increased by 1.41 folds (AOR = 1.41[1.10–1.81], p-value = 0.007) compared to those with higher ionised calcaemia. Total calcaemia and albuminaemia were not found to have any effect. Concerning the HC and maternal BMI immediately after birth, none of the factors had a statistically significant relationship with any of the three factors.

Table 3 shows the outcomes associated with calcium supplementation in pregnancy. Calcium supplementation was found to have non-statistically significant associations with maternal BMI immediately after birth, hypertension in pregnancy, LBW, LBL, LBC, and HC. However, calcium supplementation had a statistically significant association with the AS at the first and fifth minutes. Women who took calcium supplements in pregnancy had their odds of having babies with the first-minute AS < 7 reduced by 0.42 folds (OR = 0.42[0.24–0.72], p-value = 0.002). Also, women who took calcium supplements in pregnancy had their odds of having babies with a fifth-minute AS < 7 reduced by 0.31 folds (AOR = 0.25[0.12–0.50], p-value = 0.000).

Table 3

Association of calcium supplementation in pregnancy with maternal and foetal outcomes.

Outcomes	Odds Ratio	p-value
Maternal body mass index immediately after birth above 25 kg/m2	1.16[0.87–1.56]	0.296
Hypertension in pregnancy	0.91[0.59–1.41]	0.672
Low birth weight (< 2500g)	0.71[0.43–1.17]	0.174
Low birth length (≤ 48cm)	0.89[0.58–1.39]	0.626
Brachial circumference ≤ 11cm	1.22[0.92–1.63]	0.160
Head circumference ≤ 35cm	1.05 [0.80–1.39]	0.704
Apgar score at first minute < 7	0.42 [0.24–0.72]	0.002*
Apgar score at fifth minute < 7	0.25 [0.12–0.50]	0.000*

*Statistically significant

Discussion

This study evaluates the relative effect of total calcaemia, ionised calcaemia, albuminaemia and calcium supplementation on some key obstetric outcomes. The prevalence of LBW, macrosomia, and hypertension in pregnancy was 6.27 [4.97–7.89]%, 4.78 [3.65–7.89]%, 10.24 [8.57–12.20]%, respectively. Following multiple logistic regression, women with iCa levels ≤ 1.31mmol/L had significantly increased odds of hypertension in pregnancy (AOR = 2.47 [1.63–3.74], p-value = 0.000), having babies with LBW (AOR = 2.02[1.33–3.61], p-value = 0.002), LBL (AOR = 2.00 [1.34–2.99], p-value = 0.001), LBC (AOR = 1.41[1.10–1.81], p-value = 0.007), first minute AS < 7 (AOR = 3.08[1.70–5.59], p-value = 0.000) and fifth minute AS < 7 (AOR = 2.86[1.32–6.16], p-value = 0.007). Ionised calcaemia was not significantlt associated with maternal BMI immediately after birth and the HC of the baby. Total calcaemia was found to have no association with any of the selected outcomes while women with albuminemia ≤ 30mg/L had increased odds of having LBW babies (AOR = 3.40[1.96–5.91], p-value = 0.000), and babies with AS ≤ 7 at the first minute (AOR = 2.07[1.16–3.70], p-value = 0.013). Calcium supplementation was not associated with any of the selected outcomes except for the first (OR = 0.42[0.24–0.72], p-value = 0.002) and fifth minute AS (OR = 0.25[0.12–0.50], p-value = 0.000).

According to the WHO, a baby has LBW if born with a weight below 2500g, irrespective of the gestational age [31, 32]. LBW is responsible for 60–80% of neonatal deaths in developing countries [28]. Our study included women at term (at least 37weeks complete weeks) and had a prevalence of LBW at term of 6.27%. As expected, this is smaller than the prevalence of 13.5% reported in Buea and the 11% national prevalence [28], which considered deliveries irrespective of gestational age. The prevalence in this study corresponds more to the proportion of babies who might have experienced intra-uterine growth restriction or, better still, were just small for their gestational age babies at term [33, 34].

On the other hand, the prevalence of macrosomia was 4.78 [3.65–7.89]%, which is lower than the 6.41% reported in Cameroon about two decades ago [35]. This 6.41% is, however, found in our reported CI. This is also smaller than the findings in a recent study in Buea, which reported a prevalence of macrosomia of 9.5% [36]. These discrepancies may be due to changes in nutritional behaviours in pregnancy and the evolving quality of antenatal care over the years. Macrosomia has been associated in literature with labour dystocia, increased likelihood of severe perineal tears, higher caesarean section rate and instrumental delivery, postpartum haemorrhage, poor Apgar scores at the fifth minute and neonatal death [37-39].

According to our results, 10.24% had hypertensive disorders in pregnancy. Hypertensive disorder in pregnancy is a generic term that includes pre-existing and gestational hypertension, pre-eclampsia and pre-eclampsia with convulsions (eclampsia) [40, 41]. Hypertensive disorders in pregnancy remain a significant cause of maternal and foetal morbi-mortality [42], standing out as the second leading cause of maternal mortality worldwide [43]. The prevalence of hypertensive diseases in pregnancy recorded in this study is similar to the global prevalence of 10% [44].

Several studies have evaluated the relationship between maternal blood calcium levels and hypertension in pregnancy. However, studies that considered the effect of iCa are rare. Following multiple logistic regression, women with iCa levels ≤ 1.31mmol/L had their odds of hypertension in pregnancy increased by 2.47 folds (AOR = 2.47[1.63–3.74], p-value = 0.000). In a study in which tCa was used, the mean total calcaemia among women with pregnancy-induced hypertension was significantly lower than that in women with normal pregnancy [16]. Also, in a very recent Ethiopian study, similar findings have been reported, and in addition, pre-eclampsia was found to have a stronger association with iCa compared with tCa [19]. However, contrary to reports in this recent study, we did not find total calcaemia to influence the likelihood of hypertension in pregnancy. Our results suggest that calcium intake and supplementation in regulating blood pressure in pregnancy do so by affecting the ionised fraction of calcium in blood. According to the pathophysiology, low calcium levels induce the production of the parathyroid hormone, which in turn leads to increased intracellular calcium concentrations and subsequent vasoconstriction, thereby increasing blood pressure [19]. This study stands out as one of the pioneer contributions that evaluate the relative impact of ionised and tCa on blood pressure values in pregnancy. The recent study carried out in Ethiopia was focused only on pre-eclampsia, while this study considers hypertension in pregnancy as a block.

In this study, calcium supplementation was not found to affect ionised and tCa levels, even though tCa levels were positively associated with iCa levels (r = 26.12, p-value = 0.000). The same non-significant relationship between calcium intake and tCa has been reported in India [14] and Italy [45]. Similar findings have been found in a recent Ethiopian study that evaluated both total and ionised calcaemia [19]. These results suggest that calcium supplementation might not be associated with lower blood pressure values in pregnancy or contributes to reducing blood pressure not by increasing total calcaemia, but by maintaining a stable concentration of ionised calcaemia.

Multiple studies have reported that total calcaemia in pregnancy has no effect on FBW [7, 46, 47]. In line with these findings, our results found no association between total calcaemia and FBW. However, we found ionised calcaemia to be a strong potential factor that affects FBW. Following multiple logistic regression, women with iCa levels ≤ 1.31 mmols/L had their odds of having babies with LBW increased by about 2 folds (AOR = 2.19[1.33–3.61], p-value = 0.002). Our findings were difficult to compare, given that this was practically the first evaluation of this association. This can be explained by the inevitable intervention of calcium signalling in the different growth and differentiation pathways [48]. In addition, better iCa concentrations in maternal blood will mean more calcium available to cross to the baby for bone development. Calcium supplementation did not show a statistically significant association with FBW. Likely, calcium supplementation does not affect FBW. This has been reported by other studies and systematic reviews [49, 50]. However, contrasting evidence has suggested beneficial effects of increased calcium intake in reducing the likelihood of LBW [51, 52].

Serum albumin levels have been described as a marker protein for nutritional status [53]. In our study, women with albumin levels ≤ 30 mg/L had their odds of having babies with LBW increased by 3.40 folds (AOR = 3.40[1.96–5.91], p-value = 0.000). Here, low albumin is likely not to be a direct cause of LBW but might be a marker of the limited availability of amino acids for the synthesis of different structural proteins of the baby, hence the effect on FBW. Similar results have been found in Japan [54] and Nigeria [53].

Moreover, significant associations were found between iCa levels and other parameters like the BL and BC. Following multiple logistic regression, women with iCa levels ≤ 1.31mmol/L had their odds of LBL in the babies increased by 2 folds (AOR = 2.00[1.34–2.99], p-value = 0.001). Also, women with iCa levels ≤ 1.31mmol/L had their odds of LBC (≤11cm) in the baby increased by 1.41 folds (AOR = 1.41[1.10–1.81], p-value = 0.007). To the best of our knowledge, no studies have evaluated this relationship and compared the effect of ionised and tCa on these outcomes. There is, therefore, no data for comparison. These observations can be explained by the interference of available iCa in signalling pathways and the consequential availability of diffusible calcium for foetal bone development. These outcomes are dependent on iCa and not the tCa. In a prospective cohort study, high parathyroid hormone with low 25(OH)D or very little calcium intake was significantly associated with LBL and lower HC [55]. However, the direct influence of calcium and the different fractions was not evaluated in this study.

Our study associated higher ionised serum calcium levels with better foetal outcomes in terms of the AS at the first and fifth minute. We did not find any studies evaluating the effect of iCa levels on the AS and therefore could not compare our results. However, better concentrations of the physiologically active fraction of calcium should be associated with a better physiological response of the baby at birth. A nutritional state marker like albumin was found to significantly predict the AS at birth. Lower albumin concentrations could mean limited protein nutrients and amino acids required for the normal foetal response at birth. Albumin might not be directly involved but could be an explorable marker of maternal nutritional state and, therefore, a good predictor of foetal response at birth. Calcium supplementation could directly improve the ionised calcaemic poll required for the response at birth. This explains the association observed between calcium supplementation in pregnancy and AS. Similar results have been found in literature showing that calcium supplementation in pregnancy is significantly associated with a reduced likelihood of having babies with fifth-minute AS < 7 [56].

Limits of the study

The associations established in this write up should be taken with care. The cross-sectional nature of this research only allows for the emission of hypotheses and not causal relationships. Calcium concentrations were measured only once, and therefore acute variations could not be differentiated from chronic variations. Moreover, our study evaluated outcomes only at term and birth. Even though adverse maternofoetal outcomes associated with hypocalcaemia are expected to have effects on neonatal health, our study did not assess the effect of maternal calcaemic states on neonatal health at key periods of neonatal follow-up. Notwithstanding, the relationships established in this manuscript are pertinent, original, even if they require further investigations with more adapted study designs. Our research stands out as a pioneer study in evaluating the effect of iCa, tCa, and albumin on foetal outcomes.

Conclusion

The prevalence of LBW, macrosomia and hypertension in pregnancy was 6.27 [4.97–7.89]%, 4.78 [3.65–7.89]%, 10.24 [8.57–12.20]%, respectively. Maternal iCa levels ≤ 1.31mmol/L significantly increase the odds of having babies with LBW, LBL, LBC, low AS at the first and fifth minute and maternal hypertension in pregnancy. Low maternal albuminemia is significantly associated with LBW and AS < 7 at the first minute. Maternofoetal outcomes do not depend on total calcaemia, given that it does not affect BW, hypertension in pregnancy, AS, BL, BC, and BMI immediately after birth.

Even though iCa levels remain relatively normal in normal pregnancies, it remains the strongest predictor of foetal outcomes. Calcium supplementation in pregnancy does not significantly affect these outcomes, but the direction of influence ties with observations of calcaemic effects. Notwithstanding, calcium supplementation in pregnancy significantly affects the AS at the first and fifth minutes. Routine pregnancy follow-up should include routine evaluation of maternal calcaemic states, particularly the ionised fraction, to detect the low-normal concentrations likely to impact maternal and foetal outcomes. The effect of ionised calcaemia on maternofoetal outcomes suggests a revisiting of the normal cut-offs for ionised calcaemia in pregnancy.

Data base of determinants and effects of low serum calcium in pregnancy.

(ACCDB)

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21 Jan 2022

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: No

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Reviewer #1: No

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6. Review Comments to the Author

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Reviewer #1: Review comments on PONE-D-21-33966

Major comments

This is an article discussing an association between pregnant maternal serum calcium, ionized calcium and fetal outcomes including anthropometric indecies and physiological status using Apgar scores (AS) at on and five minutes after birth. The authors successfully showed an association of ionized calcium and low birth weight, low AS scores, and brachial circumferences among 1,074 pregnant women. However, the reason why the authors pay attention to serum calcium and fetal growth and no information how amount of calcium supplement or calcium daily intake had during each trimester as the authors discussed in the discussion part. Moreover, they did not show mother’s nutritional status including BMI at baseline and body weight gain during pregnancy. Unless such information is not shown, clinicians could not identify who is at risk of low ionized calcium concentration from mother’s nutrition. Additionally, English writing seems poor to be edited by native English writers. The timing to take blood also must be shown whether before breakfast, fating period, or early morning to standardize serum calcium concentrations.

In conclusion, I feel that this article must show mother’s nutritional status and daily nutrients intake in whom serum ionized calcium is lower than cutoff value and English edits. However, the authors’ eye point of ionized calcium with potential function of fetal cell development seems opening novel scientific door to faeto-maternal affect. As my conclusion, I must ask major revision for the points above mentioned. Otherwise, this must be rejected.

Minor comments

1. No explanations of abbreviations in all tables.

2. Why they measured brachial circumference? I feel this must be overestimated because of effect of long-term floating in yolk sac fluid before birth.

3. What contents of interview questionnaire and was it validated, although the authors described that questionnaires were validated but no references to explain that?

4. Line 212, “was” was duplicated in a single sentence.

5. The part of the end of discussion must be divided to “Limitations of the study” like the other articles.

6. Line 387: the word of “cause-effect relationship” might be changed to causal association.

**********

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Reviewer #1: Yes: Teruyoshi Amagai, MD, PhD

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28 Jan 2022

Dear Editor/Reviewer,

We wish to thank you for the contributions you have been put in to strengthen this manuscript. We have taken time and gone through each point and have responded while making changes to the manuscript. The response to reviewers comments have been presented here in a tabular form (below). Also, the recommendations of the editor in terms of PLoS one guidelines, ethical statement and data availability statement have been addressed.

REVIEWER’S COMMENTS

This is an article discussing an association between pregnant maternal serum calcium, ionized calcium and fetal outcomes including anthropometric indecies and physiological status using Apgar scores (AS) at on and five minutes after birth. The authors successfully showed an association of ionized calcium and low birth weight, low AS scores, and brachial circumferences among 1,074 pregnant women.

Resp: Thank you for your observation.

However, the reason why the authors pay attention to serum calcium and fetal growth and no information how amount of calcium supplement or calcium daily intake had during each trimester as the authors discussed in the discussion part.

Resp: Thanks for this observation. We designed and developed a research protocol which we submitted as was published in PLoS One (1). The present manuscript provides a report of some of the research findings. While we do acknowledge the limits associated with the cross sectional design (stated in the limits), we seek to faithfully report the outcomes of the study as designed in the protocol and recommended by PLoS One editorial policies on open science. This work has been divided into three major write ups which have all been submitted to PLoS One.

We have a manuscript evaluating calcium intake (in terms of calcium supplementation practices in pregnancy) and determines factors affecting this practice in pregnancy (given that it is recommended by the WHO to be systematic in LMIC). This was submitted on the 1st 0ct 2021 (PONE-D-21-31679).

The second manuscript from this registered protocol focused on evaluating the prevalence of ionised and total hypocalcaemia and associated socio-demographic, obstetric and nutritional factors using standard statistics. It also finds out how calcium supplementation variables like daily dose, duration, taking calcium with other supplements affect calcaemic states. This has already gone through revision (PONE-D-21-31669R1)

The present manuscript therefore targets the hypocalcaemia-associated outcomes. It evaluates the prevalence of some adverse maternofoetal outcomes and finds out associations between calcaemic states (ionised and total calcium), albumin and maternofoetal outcomes. We feel that mixing calcium intake/calcium supplementation into this manuscript does not help us respond to any of its objectives. This aspect has been dealt with in our first manuscript.

Moreover, they did not show mother’s nutritional status including BMI at baseline and body weight gain during pregnancy. Unless such information is not shown, clinicians could not identify who is at risk of low ionized calcium concentration from mother’s nutrition. Resp: We thank the Reviewer for this point. As stated in the in the registered protocol, we were interested in evaluating nutritional habits or behaviours and how they influence calcaemic states (already addressed in the second manuscript). We acknowledge that the nutritional status of these women was not evaluated in our protocol.

As stated in the registered protocol, our study was cross sectional in design and did not aim at finding causative factors of low hypocalcaemia in pregnancy (which would ideally be done using a longitudinal design). Evaluation of risk factors associated with hypocalcaemia is addressed by the second manuscript.

We used a crude BMI which was gotten immediately after delivery. Measurement of overweight and obesity in pregnancy using BMI was not one of our objectives. We believed that the error associated with its evaluation immediately after birth was common in all included women and the trend could therefore still be useful.

We understand that criticisms of this sort greatly improves the study and lends another dimension of its utility. We have taken note of this and will incorporate the pertinent points in subsequent research on this important subject

Additionally, English writing seems poor to be edited by native English writers.

Resp: Thanks for your observation. The whole manuscript has been edited by a native English speaker.

The timing to take blood also must be shown whether before breakfast, fating period, or early morning to standardize serum calcium concentrations.

Resp: Thanks for your pertinent observation. Blood sample collection time for calcium assays was random and depended on the time at which the participant was included into the study as has been the case in similar studies (2–6). No particular fasting indications were given to participants before sample collection. However, as stated in the methods section, precautions were taken to make sure measured calcium levels best reflect the calcaemic states of the participants.

In conclusion, I feel that this article must show mother’s nutritional status and daily nutrients intake in whom serum ionized calcium is lower than cutoff value and English edits.

Resp: We want to thank the Reviewer for the time taken to assess the value of this study, which he greatly appreciates and therefore provided constructive feedback. We have taken note of some of the pertinent comments which, unfortunately, can only be adequately addressed in a new study (longitudinal). We plan to take these into consideration as part of our future study.

However, the authors’ eye point of ionized calcium with potential function of fetal cell development seems opening novel scientific door to faeto-maternal affect.

Resp: Thank you very much.

As my conclusion, I must ask major revision for the points above mentioned. Otherwise, this must be rejected.

Resp: Thanks for your recommendation. We have provided a point by point response to the comments and substantially revised the manuscript. We remain open should there be other issues to address prior to publication of the manuscript following the publication of its protocol.

MINOR REVIEWER’S COMMENTS RESPONSES

1. No explanations of abbreviations in all tables.

Resp: Thanks for your observations, the tables have been edited and all abbreviations removed. See table 1, 2 and 3.

2. Why they measured brachial circumference? I feel this must be overestimated because of effect of long-term floating in yolk sac fluid before birth.

Resp: Thanks for the pertinent point. We agree with the Reviewer that this measurement might be overestimated. But our aim was not to define with exactitude the prevalence of low brachial circumference at birth. In addition, we needed the brachial circumference at birth which is standardly measured like that in the hospitals (7,8). Moreover, even with the error, the trend of variation with calcaemic states will still be detected (7,8).

3. What contents of interview questionnaire and was it validated, although the authors described that questionnaires were validated but no references to explain that?

Resp: Thanks for your observations. The questionnaire used in this study was published with the registered protocol which has been cited in the manuscript. The sentence fraction “validated questionnaires” is only used in the first sentence of the data analysis section and describes retained questionnaires. To eliminate the ambiguity, the word has been replaced with “retained”. See page 9, paragraph 1.

4. Line 212, “was” was duplicated in a single sentence.

Resp: Thanks for the observation. The error has been corrected. See line 217, paragraph 2, page 11.

5. The part of the end of discussion must be divided to “Limitations of the study” like the other articles.

Resp: Thanks for your recommendation. The section “limits of the study” has been added. See paragraph 1, page 21

6. Line 387: the word of “cause-effect relationship” might be changed to causal association.

Resp: Thanks for your suggestion. It has been changed to causal. See paragraph 1, page 21

References

1. Ajong AB, Kenfack B, Ali IM, Yakum MN, Aljerf L, Telefo PB. Hypocalcaemia and calcium intake in pregnancy: A research protocol for critical analysis of risk factors, maternofoetal outcomes and evaluation of diagnostic methods in a third-category health facility, Cameroon. PLoS One. 2020;15(11 November).

2. Ajong AB, Kenfack B, Ali IM, Yakum MN, Telefo PB. Prevalence and correlates of low serum calcium in late pregnancy: A cross sectional study in the Nkongsamba Regional Hospital; Littoral Region of Cameroon. PLoS One. 2019;14(11).

3. Bako B, El-Nafaty AU, Mshelia DS, Gali RM, Isa B, Dungus MM. Prevalence and risk factors of hypocalcemia among pregnant and non-pregnant women in Maiduguri, Nigeria: A cross-sectional study. Niger J Clin Pract. 2021;

4. Gebreyohannes RD, Abdella A, Ayele W, Eke AC. Association of dietary calcium intake, total and ionized serum calcium levels with preeclampsia in Ethiopia. BMC Pregnancy Childbirth. 2021;21(1).

5. Kumar A, Agarwal K, Devi SG, Gupta RK, Batra S. Hypocalcemia in Pregnant Women. Biol Trace Elem Res. 2010 Jul;136(1):26–32.

6. Benali AI, Demmouche A. Calcium Deficiency among Pregnant Women and their Newborns in Sidi Bel Abbes Region, Algeria. J Nutr Food Sci. 2014;04(06):4–7.

7. Shankar R, Ramarajan A, Rani S, Seshiah V. Anthropometric and Skin Fold Thickness Measurements of Newborns of Gestational Glucose Intolerant Mothers: Does it Indicate Disproportionate Fetal Growth? J Obstet Gynecol India. 2020;

8. Tiruneh C, Teshome D. Prediction of Birth Weight by Using Neonatal Anthropometric Parameters at Birth in Finote Selam Hospital, Ethiopia. Pediatr Heal Med Ther. 2021;

Submitted filename: RESPONSE TO REVIEWERS.docx

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4 Apr 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Linglin Xie

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript adhere to the experimental procedures and analyses described in the Registered Report Protocol?

If the manuscript reports any deviations from the planned experimental procedures and analyses, those must be reasonable and adequately justified.

Reviewer #1: No

Reviewer #2: Yes

**********

2. If the manuscript reports exploratory analyses or experimental procedures not outlined in the original Registered Report Protocol, are these reasonable, justified and methodologically sound?

A Registered Report may include valid exploratory analyses not previously outlined in the Registered Report Protocol, as long as they are described as such.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Are the conclusions supported by the data and do they address the research question presented in the Registered Report Protocol?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. The conclusions must be drawn appropriately based on the research question(s) outlined in the Registered Report Protocol and on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Reviewer’s comments on PLOE-D-21-33966 R1

After the authors revised an article entitled:

An article seems more sophisticated and scientific. However, its devised version does not change the results and their clinical meanings to improve outcomes on maternal and fetal health. The authors found that ionized Ca of the pregnant mothers seems to have impacts on fetal outcomes, including Apgar scores at one and five minutes after birth. However, the issues of neonates must be occurrence of events seen at longer observational periods of time, such as 7-day, 14-day, or 28-day mortality or IRDS. If neonates have lower Apgar score (AS) at birth, neonatologists could treat them properly and AS seems no effects on 7-, 14-,28-days mortality.

In conclusion, although the authors had tremendously a lot of time to study, the results seems no clinical impacts on neonates and this might be rejected.

Reviewer #2: All comment raised by the first and second revision are addressed:

Thanks for that and congratulation.

**********

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Reviewer #1: No

Reviewer #2: No

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9 Apr 2022

RESPONSE TO REVIEWER’S COMMENTS

We wish to appreciate once more the efforts of the editor and the reviewers to improve the quality of this manuscript. We have presented the response to each reviewer’s comment in the table below.

Comments and Response

1. Does the manuscript adhere to the experimental procedures and analyses described in the Registered Report Protocol?

If the manuscript reports any deviations from the planned experimental procedures and analyses, those must be reasonable and adequately justified.

Reviewer #1: No

Reviewer #2: Yes

Resp: Thank you for your response. Our manuscript partly adheres to experimental procedures and analyses reported in the registered protocol. All the data collection steps were respected from the number of participants, recruitment of participants, methods of biochemical assays, measurement of maternal foetal parameters and data analysis. No additional data were collected to be able to tackle the objectives set in this manuscript.

We reported a slight deviation in the methods where we explained why instead of considering only the Nkongsamba Regional Hospital, we had to include 4 major health facilities in the Nkongsamba Health District. This is explained in the manuscript with the reason. Also, instead of excluding a set of participants with hypocalcaemia-causing pathologies, we including all women who were apparently healthy in the study and this statement was included and justified. See methods,line 121-132 and line 138-141. Page 8.

2. If the manuscript reports exploratory analyses or experimental procedures not outlined in the original Registered Report Protocol, are these reasonable, justified and methodologically sound?

A Registered Report may include valid exploratory analyses not previously outlined in the Registered Report Protocol, as long as they are described as such.

Reviewer #1: Partly

Reviewer #2: Yes

Resp: Thank you for your response. This manuscript was written out from one of the objectives of the registered protocol.

Objective 3: Determine the prevalence of hypertensive disorders in pregnancy among hypocalcaemic pregnant women in the NRH and describe hypocalcaemia-associated maternofoetal outcomes (with foetal outcomes evaluated assecondary outcomes) (1). We reformulated this objective to that described in the manuscript because of the links between albumin, total and ionised calcaemia. We were interested in finding out if associations existed between these factors and maternofoetal outcomes and if yes, which factors had the strongest association. This has been explained with the reason/justification in the last paragraph of the introduction. See page 6, line 111-115.

However, this did not affect any initial methods related to data collection reported in the original protocol. We did not collect any additional data to respond to the objectives of this manuscript.

As stated in the original protocol, we have also included in this revised version the prevalence of hypertension in pregnancy among hypocalcaemic women. See data analysis section, page 11, line 194-196 and results section, page 13, line 233-239.

3. Are the conclusions supported by the data and do they address the research question presented in the Registered Report Protocol?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. The conclusions must be drawn appropriately based on the research question(s) outlined in the Registered Report Protocol and on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Resp: Thank you for your answer. The conclusions of this manuscript respond to objectives of the original research protocol. These objectives were just adapted to make more meaning. The conclusions of this manuscript are based on the objectives of the manuscript.

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Resp: Thanks for your answer. We made all data underlying the findings in this manuscript fully available by submitting the data base with the previous revision.

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Resp: Thanks for your answer. The manuscript has been read and edited by a native English speaker.

Review comments to the authors

Specific Reviewer 1 comments

Comment: An article seems more sophisticated and scientific.

However, its devised version does not change the results and their clinical meanings to improve outcomes on maternal and fetal health.

Resp: Thanks very much for your observation. We hope that our explanations help clarify some issues

Comment: The authors found that ionised Ca of the pregnant mothers seems to have impacts on fetal outcomes, including Apgar scores at one and five minutes after birth. However, the issues of neonates must be occurrence of events seen at longer observational periods of time, such as 7-day, 14-day, or 28-day mortality or IRDS. If neonates have lower Apgar score (AS) at birth, neonatologists could treat them properly and AS seems no effects on 7-, 14-,28-days mortality.

Resp: Thank you very much for your comment. Our manuscript presents results from a registered protocol. We perfectly agree that neonatal follow up periods extend to 28days, and our study did not look as far as that to see if other low calcium-related adverse outcomes could occur. So long term effects of low calcium were not our target (this has been included as a limit of this study). See page 25, line 421-424.

According our methods, the factors (were low calcium, low albumin and calcium supplementation in the mother), and our outcomes of interest were measured at term and birth. All selected foetal outcomes have been shown in literature to have potential negative effects on the health of the child in future. For example, low Apgar scores have been associated with poor neurological development and lower IQ (2,3), respiratory distress, hypothermia, feeding problems (4), higher neonatal mortality (5). Similar effects can be cited for low birth weight and others.

More interest is put on which maternal variables could be acted upon to reduce adverse maternal and foetal outcomes. We found out that the key determinant was the ionised calcaemic state, sometimes the calcium supplementation.

Children with poor Apgar scores could be resuscitated by neonatologist as stated by the reviewer and the course of evolution might not show any long term effects. But we feel that detecting a factor like the calcaemic state of the mother (and acting on it) could help reduce the number of children born with low Apgar score. The same goes for children with low birth weight (responsible for 60-80%) of neonatal deaths (6). Preventing the birth of babies with low Apgar scores and low birthweight is, therefore, better than having neonatologists ready to resuscitate these babies.

Moreover, our finding of the effect of ionised calcaemic state on hypertensive disease in pregnancy is a turning point. Acting only at this level prevents its occurrence and adverse maternal and foetal outcomes associated with hypertension in pregnancy (especially preeclampsia and eclampsia).

Comment: In conclusion, although the authors had tremendously a lot of time to study, the results seems no clinical impacts on neonates and this might be rejected.

Resp: Thanks for your comment. We hope that our explanations have convincing enough. Do maternal variables like calcaemic state, albuminaemia state and calcium supplementation affect maternal and foetal outcomes? Can these factors be acted upon to prevent the occurrence of adverse outcomes? We think the answer is yes. In this light, we believe our findings can contribute to improving maternal and feotal health.

Reviewer 2 comments

All comments raised by the first and second revision are addressed:

Thanks for that and congratulation.

Resp: Thank you very much for your valuable contributions

References

1. Ajong AB, Kenfack B, Ali IM, Yakum MN, Aljerf L, Telefo PB. Hypocalcaemia and calcium intake in pregnancy: A research protocol for critical analysis of risk factors, maternofoetal outcomes and evaluation of diagnostic methods in a third-category health facility, Cameroon. PLoS One. 2020;15(11 November).

2. Hassen TA, Chojenta C, Egan N, Loxton D. The association between the five-minute apgar score and neurodevelopmental outcomes among children aged 8−66 months in Australia. Int J Environ Res Public Health. 2021;18(12).

3. Tweed EJ, Mackay DF, Nelson SM, Cooper SA, Pell JP. Five-minute Apgar score and educational outcomes: Retrospective cohort study of 751 369 children. Arch Dis Child Fetal Neonatal Ed. 2016;101(2):F121–6.

4. Thavarajah H, Flatley C, Kumar S. The relationship between the five minute Apgar score, mode of birth and neonatal outcomes. J Matern Neonatal Med. 2018;31(10):1335–41.

5. Cnattingius S, Norman M, Granath F, Petersson G, Stephansson O, Frisell T. Apgar Score Components at 5 Minutes: Risks and Prediction of Neonatal Mortality. Paediatr Perinat Epidemiol. 2017;31(4):328–37.

6. Njim T, Atashili J, Mbu R, Choukem SP. Low birth weight in a sub-urban area of Cameroon: An analysis of the clinical cut-off, incidence, predictors and complications. BMC Pregnancy Childbirth. 2015;15(1).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

2 May 2022

PONE-D-21-33966R2

How do ionised calcium, total calcium, total albumin, and calcium supplementation affect maternofoetal outcomes? analysis from a resource-limited setting

PLOS ONE

Dear Dr. Ajong,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Kind regards,

Linglin Xie

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript adhere to the experimental procedures and analyses described in the Registered Report Protocol?

If the manuscript reports any deviations from the planned experimental procedures and analyses, those must be reasonable and adequately justified.

Reviewer #1: Yes

**********

2. If the manuscript reports exploratory analyses or experimental procedures not outlined in the original Registered Report Protocol, are these reasonable, justified and methodologically sound?

A Registered Report may include valid exploratory analyses not previously outlined in the Registered Report Protocol, as long as they are described as such.

Reviewer #1: Yes

**********

3. Are the conclusions supported by the data and do they address the research question presented in the Registered Report Protocol?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. The conclusions must be drawn appropriately based on the research question(s) outlined in the Registered Report Protocol and on the data presented.

Reviewer #1: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Reviewer’s comment on PONE-D-21-33966-R2

Major comments

This is revised article studying an association between adverse events of neonate and biochemical parameters of serum calcium and albumin concentration of pregnant mothers at 37 and over of pregnant period before delivering neonates. The authors found associations of maternal lower ionized calcium level <= 131 mmol/L and low birth weight, low birth length, low brachial circumference at birth, and low Apgar score at one and fifth minutes after birth. Moreover, they stated that calcium supplementation significantly improved the Apgar scores at the first and fifth minutes showing their results in Table 3. Form reading carefully their descriptions about evaluating maternal calcium supplementations during pregnant periods, it is unclear how much thy took calcium supplementation adding on dietary intake. The methodological problem that I am feeling is that daily total calcium intakes at late pregnant periods of meal and calcium supplementation must be calculated. Otherwise, it must be unclear which pregnant mother must be target to supplement calcium because she is deficient calcium intake. In addition, serum vitamin D concentrations and time durations of sunlight exposure also must be considered when which pregnant women is the target of calcium supplementations to prevent poor maternal or neonatal outcomes. Without disclosed above mentioned problems must be resolved, this article could not provide meaningful information to health care professionals.

In conclusion, I would say that this revised article must not be accepted.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Teruyoshi Amagai, MD, PhD, University of Jikei Health Care Sciences, Osaka, Japan

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For journal use only: PONEDEC3

12 May 2022

Response to Reviewers

Comment: This is revised article studying an association between adverse events of neonate and biochemical parameters of serum calcium and albumin concentration of pregnant mothers at 37 and over of pregnant period before delivering neonates. The authors found associations of maternal lower ionized calcium level <= 131 mmol/L and low birth weight, low birth length, low brachial circumference at birth, and low Apgar score at one and fifth minutes after birth. Moreover, they stated that calcium supplementation significantly improved the Apgar scores at the first and fifth minutes showing their results in Table 3.

Response: Thank you for the review. Our article evaluates associations between hypocalcalcaemic states and maternofoetal outcomes. We have reviewed the title to fit the design of the study. In essence, our study evaluated associations which could be potential hypotheses for future longitudinal studies (for cause-effect relationships). This is recognized as a limit to our findings in the manuscript. See title and section on limits.

Comment: Form reading carefully their descriptions about evaluating maternal calcium supplementations during pregnant periods, it is unclear how much thy took calcium supplementation adding on dietary intake. The methodological problem that I am feeling is that daily total calcium intakes at late pregnant periods of meal and calcium supplementation must be calculated. Otherwise, it must be unclear which pregnant mother must be target to supplement calcium because she is deficient calcium intake. In addition, serum vitamin D concentrations and time durations of sunlight exposure also must be considered when which pregnant women is the target of calcium supplementations to prevent poor maternal or neonatal outcomes. Without disclosed above mentioned problems must be resolved, this article could not provide meaningful information to health care professionals.

In conclusion, I would say that this revised article must not be accepted.

Response: Thank you for your concern. Our objective in this manuscript was not to evaluate the calcium intake of the participants in the diet and supplements. As stated in response to reviewers 1, We have a manuscript evaluating calcium intake (in terms of calcium supplementation practices in pregnancy) and determines factors affecting this practice in pregnancy (given that it is recommended by the WHO to be systematic in LMIC for the prevention of hypertensive diseases in pregnancy). This was submitted on the 1st 0ct 2021 (PONE-D-21-31679).

Our key problem was to evaluate the relative strength of association that ionized and total calcaemic states could have on maternofoetal outcomes. In doing this we attached a simple question to see if there was an association between “haven taken calcium supplements in pregnancy” and maternofoetal outcomes. We were not looking for which women to supplement and which women not to supplement.

We understand that Vit D concentrations and exposure to sunlight contribute to providing active calcitriol for intestinal absorption of calcium. Vit D and sunlight exposure concur to increasing concentrations of calcitriol which increases intestinal calcium absorption. Therefore, we believe that to evaluate the association between calcaemic states and maternofoetal outcomes, there is no need to control for sunlight exposure and Vit D concentrations. This because the terminal point of Vit D concentrations and sunlight exposure is calcaemia.

Submitted filename: Response to Reviewers R3.docx

Click here for additional data file.

5 Jul 2022

Adverse maternofoetal outcomes associated with ionised calcaemia, total calcaemia, albuminaemia, and calcium supplementation in pregnancy: analysis from a resource-limited setting

PONE-D-21-33966R3

Dear Dr. Ajong,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Dylan A Mordaunt, MD, MPH, FRACP

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for your submission. I have taken over as academic editor and have familiarised myself with the previous reviews and decisions. Fundamentally, the recommendations have been addressed by the reviewers and the manuscript now meets PLoS One's criteria for publication. Some of the somewhat unclear comments from the reviews do not directly relate to PLoS One's criteria for publication, and the manuscript reflects execution of a peer reviewed protocol.

With specific reference to the criteria for publication:

1. The study appears to present the results of original research. Reviewer comments seem to address the perceived value of the research and a fixed perspective on limitations rather than interpreting the data in the context of an a priori agreed protocol.

2. Results reported do not appear to have been published elsewhere.

3. Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail. Again, review comments do not detract from this and indeed the protocol was peer reviewed and published in PLoS One. The stated limitations are a lack of presentation of data not collected. Momentary ionized calcium is not primarily a reflection of dietary calcium, so I'm not convinced this is a critical limitation, and wasn't found to be in the peer-reviewed protocol.

4. Conclusions are presented in an appropriate fashion and are supported by the data.

5. The article is presented in an intelligible fashion and is written in standard English. There are some "turns of phrase" or possibly idioms that I wasn't familiar with, such as "inexistent", but these appear to be accepted synonyms and reflect international dialects of English rather than poor fluency.

6. The research meets all applicable standards for the ethics of experimentation and research integrity.

7. The article adheres to appropriate reporting guidelines and community standards for data availability. A report like this could benefit from reference to a standardised reporting checklist like STROBE or extensions, but the way this is structured is high quality and all the detail is there, it would be a minor value add and optional for the authors.

Reviewers' comments:

21 Jul 2022

PONE-D-21-33966R3

Adverse maternofoetal outcomes associated with ionised calcaemia, total calcaemia, albuminaemia, and calcium supplementation in pregnancy: analysis from a resource-limited setting

Dear Dr. Ajong:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Associate Professor Dylan A Mordaunt

Academic Editor

PLOS ONE