Fetal macrosomia, fetal insulin, and insulin-like growth factor- 1 among neonates in Lagos, Nigeria: A case-control study.

PURPOSE: Fetal macrosomia is associated with perinatal injuries. The purpose of this study was to assess the relationship between fetal insulin, insulin-like Growth factor-1(IGF-1), and macrosomia in a resource-limited setting.
METHOD: This was a case-control study at tertiary and secondary health facilities in Lagos, Nigeria. One hundred and fifty mother-neonate pairs were recruited, and their socio-demographic and obstetric history was recorded. Fetal cord venous blood was collected at birth, and neonatal anthropometry was measured within 24hrs of life. Insulin and IGF-1 assay were measured with Enzyme-Linked Immunosorbent Assay (ELISA). Pearson's Chi-square was used to assess the association between categorical variables and macrosomia. Spearman's rank correlation of insulin, IGF-1, and fetal anthropometry was performed. Multivariable logistic regression was used to evaluate the association of insulin and IGF-1 with fetal birth weight. A statistically significant level was set at P-value < 0.05.
RESULTS: Macrosomic neonates had mean fetal weight, fetal length, and occipitofrontal circumference (OFC) of 4.15±0.26kg, 50.85±2.09cm and 36.35± 1.22cm respectively. The median Insulin (P = 0.023) and IGF-1 (P < 0.0001) were significantly higher among macrosomic neonates as compared to normal weight babies. Maternal BMI at birth (p = 0.003), neonate's gender (p < 0.001), fetal cord serum IGF-1 (p < 0.001) and insulin assay (P-value = 0.027) were significant predictors of fetal macrosomia. There was positive correlation between cord blood IGF-1 and birth weight (r = 0.47, P-value < 0.001), fetal length (r = 0.30, P-value = 0.0002) and OFC (r = 0.37, P-value < 0.001).
CONCLUSION: Among participating mother-neonate dyad, maternal BMI at birth, neonate's gender, and fetal cord serum IGF-1 and serum insulin are significantly associated with fetal macrosomia.

Introduction

Fetal macrosomia is a significant cause of maternal and perinatal morbidity, especially in low- and middle-income countries (LMIC) [1]. Higher risk of caesarean sections, post-partum haemorrhage, birth asphyxia, neonatal trauma, and neonatal mortality are common macrosomia complications [1]. The future risk of metabolic syndrome and cardiovascular risks are linked with low birth weight, while non-diabetic obesity and cancer risks have been linked to macrosomia [2]. The negative impact of these chronic conditions on the health system and socio-economic life of LMICs is enormous. Hence, evidence of the pathophysiological processes of placental markers of fetal growth in LMICs, such as Nigeria, is required [3].

Fetal growth and development entail a complex interplay of the maternal, placenta, and fetal factors [4-6]. Pre-conceptional and antenatal maternal nutrition might impact the risk of metabolic disorders in their offspring in later life. The growth hormone-insulin-like growth factor (GH-IGF) axis is a crucial driver of fetal growth processes [3]. Maternal and fetal genetics and environmental factors contribute to birth weight in different proportions in populations [4-6]. Maternal factors, such as age, parity, weight, and gestational weight influence fetal weight [4]. Also, reports from the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC) have shown that fetal growth is influenced by fetal genes and maternal uterine-placental factors [5-7]. Therefore, an impairment of the genetic and environmental precursors of fetal growth can lead to rapid post-natal growth catch up that can cause childhood obesity and insulin resistance [5, 6].

The term “fetal macrosomia” refers to oversized fetuses. Although terms such as overweight, large for gestational age (LGA), and heavy-for-dates have been used [8, 9], none of these terms distinguishes the fetus with an abnormal body composition from normal. To make the distinction, Potter and Craig proposed the term macrosomia for fetuses with organ-weight disproportion in relation to body weight [9]. Fetal macrosomia may be symmetrical (proportionate) or asymmetrical (disproportionate). These categories are based on the ponderal index of > 2.8 (greater than 97th percentile), for asymmetric fetal macrosomia and a ponderal index of 2.2–2.8 (between the 10th and 90th percentile), for symmetric fetal macrosomia [10]. However, in this study for practical purposes macrosomia was defined as birth weight ≥ 4000 gram at term [11, 12].

Insulin is a peptide hormone synthesized by beta cells of the islets of Langerhans of the pancreas and is pivotal to regulating fat and carbohydrate metabolism in the human body. Gestational diabetes has been associated with macrosomia because of the roles of hyperinsulinism in macrosomia [13-15]. Conversely, some researchers have reported no association between cord blood insulin levels and macrosomia. Yet, insulin resistance, fetal hyperleptinaemia, and hypothalamic changes documented in the macrosomic neonates have been linked to adult life adverse outcomes [16, 17]. Insulin-like Growth Factors I and II (IGF-I; IGF-II) are polypeptides, structurally homologous to insulin, and share many biological activities [18]. Maternal and fetal IGF-1 is believed to be essential mediators in fetal growth because of its r mitogenic and metabolic actions. Maternal IGF-1 and fetal IGF-1 have been associated with macrosomia, irrespective of maternal diabetes mellitus. Higher fetal cord serum levels of IGF-1 and insulin were reported in large for gestation neonates compared with neonates appropriate for gestational age [17], with a positive correlation with fetal birth weight and other anthropometric parameters birth, including in women with malaria infection in pregnancy in Nigeria [19-22]. The differences between Caucasian and Chinese neonates on the effect of IGF-1 and fetal weight suggests a genetic influence as a higher cord serum level of IGF-1 and a significant positive association of IGF-1 with neonatal birth weight and length in neonates was seen in Caucasians [23]. Like other resource-constraint regions, sub-Saharan Africa has a high prevalence of low birth-weight neonates, yet macrosomic fetuses’ birth occurs. It is also uncertain what the contributions of IGF-1 and insulin are to fetal macrosomia in this setting. Establishing an association between IGF-1, insulin and macrosomia among Nigerian babies can aid further interventional research on the feto-maternal pathophysiology of the incidence of macrosomia and its attendant immediate maternal complications at birth and later life associated disease risks reductions. This study’s objectives were to measure fetal insulin levels and IGF-1 in macrosomic fetuses delivered at term and determine the correlation of insulin and IGF-1 with fetal anthropometric variables at birth in Lagos State, Nigeria.

Methods

This was a case-control study of mother-neonate pair at the obstetric units of four secondary and one tertiary health facilities in Lagos State. The secondary health facilities were General Hospital Surulere, General Hospital Isolo, General Hospital Mushin, Lagos Island Maternity, and the tertiary hospital was the Lagos University Teaching Hospital (LUTH), Lagos.

Mother-neonate pairs were consecutively recruited in the immediate postpartum period. Neonates were eligible if they were delivered at term (37 weeks to 42weeks gestational age). Cases were macrosomic babies (weight ≥ 4000g) (n = 100), while controls were AGA babies (n = 50). Exclusion criteria included multiple gestation, the presence of a fetal abnormality, and stillbirth.

Information on weight at the first antenatal visit, last normal menstrual period, weight at last antenatal visit, height, co-morbid states such as diabetes, hypertension, history of alcohol ingestion, parity, previous history of gestational diabetes mellitus, and previous history of macrosomic babies were obtained from the women and their medical records. The Body Mass Index (BMI) was then calculated as Weight/(Height)2. Neonatal anthropometric measurements taken within 24hrs of birth by the research team were fetal length, occipitofrontal circumference, and birth weight (using SECA 813 flat digital weighing scale, Hamburg Germany).

Fetal cord blood was collected from the placenta’s umbilical vein immediately after delivery with a 10ml syringe and placed into potassium EDTA bottles for IGF-1 and Insulin, and fluoride oxalate bottle for glucose assay. After temporary storage and transportation on Ice packs in a cooler, they were separated within a 2hrs of collection by centrifugation at 4,000 revolutions per min for 10mins to obtain the plasma. Glucose assay was performed within 6hrs of sample collection. The insulin and IGF-1 serum samples were made in aliquots into microtubes, batched, and stored in the central research laboratory at -80 0C until the recruitment conclusion. Analysis of all the samples was then conducted together.

A commercially prepared immunoassay kit (Monobind Inc. Lake Forest, CA 92630, USA), using the Enzyme-Linked Immunosorbent Assay (ELISA) method, was used to measure cord blood insulin. IGF-1 was assayed using the ELISA technique for quantitative measurement of Human IGF-1 in serum, plasma, and cell culture supernatant from Mediagnost GmbH, Aspenhaustr.25, 72770 Reutlingen, Germany (REF: E20, lot 120115). Glucose in plasma was assayed using the Trinder method. Spectrophotometry was used to measure the absorbance of the colored complex proportional to the concentration of glucose in the specimen measured at 500nm. Precision controls were used during the analysis. Within a run, within a batch, and day-to-day precision studies were carried out as required.

Approval was obtained from the Human Ethics Research Committee of the Lagos University Teaching Hospital (Ref No: ADM/DCST/HREC/APP/1810). The Heads of the General Hospitals provided permission for the study. Informed written consent was obtained from the mothers. The ethics committee approved the consent and all other aspects of the research. There was however no potential harm to the mothers and babies as this was an observational study with no intervention. Autonomy and confidentiality were maintained throughout the study.

Statistical analysis

Stata version 16 (StataCorp, Texas, USA) statistical software was used for data analysis. Continuous variables were described using Mean ± Standard deviation (SD) or median and interquartile range (IQR)–if skewed data. Categorical variables were presented as frequencies and percentages. The mean of normally distributed variables was compared across the macrosomic and AGA babies using the student’s independent t-test. Continuous variables that were not normally distributed were compared with the Mann-Whitney U test.

In contrast, Pearson’s Chi-square (of Fisher’s exact test for small numbers) was used to assess the association between categorical variables and the outcome status. Correlation of cord serum IGF-1 and Insulin with neonatal anthropometric variables (birth weight; occipitofrontal circumference; fetal length) was assessed using the Spearman’s Rank correlation coefficient. Univariable and multivariable binary logistic regression was conducted to evaluate macrosomia’s predictors with fetal IGF-1 as the primary explanatory variable. Variables with univariable P-value <0.2 was used to build the multivariable model using the backward elimination method. Some variables were chosen a priori. Similarly, two other multivariable models were built with serum insulin and glucose as the primary explanatory variable. Variables in the multivariable model I includes: IGF-1, maternal age, maternal BMI at birth, parity, gestational age, ethic group, and fetal sex. For models II and Model III, IGF-1 was replaced with insulin and glucose respectively A two-tailed test of the hypothesis was assumed, and Statistical significance was set at P-value <0.05 for all tests.

Results

One hundred and fifty mother-neonate pairs were recruited into the study from 1st June to December 2015. They comprised 100 macrosomic (birth weight ≥ 4Kg) and 50 normal weight (Birth weight < 4kg) neonates. The mean maternal age was 31.6 ±4.6 years. The mean maternal pre-pregnancy and post-partum weight was 70.5 ± 12.7Kg and 86.9±14.4Kg, respectively. Most women (98%) did not have diabetes, though 33 (22%) women had a macrosomic baby in a previous birth. There was no statistically significant difference in the characteristics of mothers of macrosomic and normal-weight babies except for Parity, Body mass index, alcohol consumption, and Previous delivery of a macrocosmic baby. Other maternal characteristics are shown in Table 1. The proportion of males (n = 59/73, 80.8%) that were macrosomic was higher than the proportion of females that were macrosomic (n = 41/77, 53.3%), P-value <0.001. For macrosomic neonates, the mean fetal weight, fetal length, and occipitofrontal circumference (OFC) was 4.2±0.3kg, 50.8±2.1cm, and 36.3± 1.2cm, respectively, while the values for normosomic neonates were 3.1±0.3kg, 47.9± 2.9cm, and 34.5±7.4cm respectively(P-value = 0.0001).

Table 1

Socio-demographic characteristics of the participants.

Characteristics	Macrosomic babies N = 100 (%)	Normal weight babies N = 50 (%)	Total N = 100 (%)	P-value	Characteristics	Macrosomic babies N = 100 (%)	Normal weight babies N = 50 (%)	Total N = 100 (%)	P-value
Maternal Age Mean (SD)	31.7 ± 4.6	32.4 ± 4.7	31.6 ± 4.6	0.6628	Pre-pregnancy BMI Mean (SD)	27.1 ±4.	24.7± 5.1	26.3 ± 4.7	0.0031*
20–24	6 (6.0)	3(6.0)	9 (6.0)	0.974	Underweight (<18.5)	0 (0.0)	2 (4.0)	2 (1.3)	0.003*
25–29	26 (26.0)	15 (30.0)	41 (27.3)		Normal weight (18.5–24.9)	35 (35.0)	30 (60.0)	65 (43.3)
30–34	37 (37.0)	16 (32.0)	53 (35.3)		Overweight (25.0–29.9)	38 (38.0)	12 (24.0)	50 (33.3)
35–39	26 (26.0)	13 (26.0)	39 (26.0)		Underweight (<18.5)	0 (0.0)	2 (4.0)	2 (1.3)	0.003*
40–44	5 (5.0)	3 (6.0)	8 (5.3)		BMI at delivery Mean (SD)	33.8 ±4.6	29.7± 5.1	32.4 ± 5.1	<0.001*
Educational qualification					Underweight (<18.5)	0 (0.0)	0 (0.0)	0 (0.0)	< 0.001*
Primary	2 (2.0)	0 (0.0)	2 (1.3)	0.737	Normal weight (18.5–24.9)	3 (3.0)	4 (8.0)	7 (4.7)
Secondary	28 (28.0)	16 (32.0)	44 (29.3)		Overweight (25.0–29.9)	17 (17.0)	24 (48.0)	41 (27.3)
Tertiary	70 (70.0)	34 (68.0)	104 (69.3)		History of Hypertension
Parity	1.5 (1–2)	0(0–2)	1(0–2)	0.0008*	Yes	4 (4.0)	1(2.00)	5 (3.3)	0.665
0	18 (18.0)	25 (51.0)	43 (28.9)	< 0.001*	No	96 (96.0)	49 (98.0)	145 (96.7)
1–4	77 (77.0)	23 (46.9)	100 (67.1)		Alcohol Consumption
Parity	1.5 (1–2)	0(0–2)	1(0–2)	0.0008*	Yes	9 (9.0)	0 (0.00)	9 (6.0)	0.030*
History of smoking					No	91 (91.0)	50 (100.0)	141 (94.0)
Yes	0 (0.00)	0 (0.00)	0 (0.00)		History of Diabetes
No	100(100.0)	100(100.0)	100(100.0)		Yes	3 (3.0)	0 (0.0)	3 (2.0)	0.551
Previous delivery of a macrocosmic baby					No	97 (97.0)	50 (100.0)	147 (98.0)
No	73 (73.0)	44 (88.0)	117 (78.0)	0.039*	Gestational age at delivery (weeks)	39.5 (39–40)	39.5 (38–40)	39.5 (38.3–40)	0.1293
Yes	27 (27.0)	6 (12.0)	33 (22.0)		Birth weight	4.2±0.3	3.07± 0.3	3.8± 0.6	< 0.001
Gender of the babies
Female	41(41.0)	36 (72.0	77 (51.3)	< 0.001
Male	59 (59.0)	14 (28.0)	73 (48.7)

SD: Standard deviation.

As shown in Fig 1, the median insulin levels were higher among macrosomic neonates, compared to the normosomic babies (4.84 (IQR: 3.1–10.1) IU/m Vs. 3.5 (2.8–6.4) IU/ml, P-value = 0.023). Similarly, the median IGF-1 level was higher among Macrosomic babies compared to Normosomic babies (85.1 (62.1–108.9) ng/ml Vs 53.8 (44.5–67.0) ng/ml, P-value < 0.0001). There was no statistically significant difference between the mean cord blood glucose levels of macrosomic and normosomic babies of 74.5 ± 30.9 mg/dl and 78.5±19.2 mg/dl, respectively (P-value = 0.4007).

Fig 1

A. Distribution of GF-1(ng/ml) among macrosomic and normosomic babies. B. Distribution of Insulin (IU/L) among macrosomic and normosomic babies.

After multivariable analysis that corrected for confounding factors, maternal BMI at birth (P-value < 0.001), fetal gender (P-value = 0.001), fetal cord serum IGF -1 (P-value = 0.002), and insulin assay (P-value = 0.034) were significantly associated with fetal macrosomia. Also, for every unit increase in IGF-1, the odds of having a macrosomic baby increased by 3% (Adjusted OR: 1.03, 95%CI: 1.01–1.05, P-value = 0.002) Table 2.

Table 2

Logistic regression of the association between insulin, insulin-like growth factor glucose and fetal macrosomia.

Variables	Unadjusted Odds Ratio (OR)	95%Confidence interval	P-value	Adjusted OR	Confidence Interval	P-value*
Insulin assay	1.05	0.99–1.11	0.078	1.07	1.01–1.1	0.034*
IGF-1	1.03	1.02–1.05	< 0.001	1.03	1.01–1.05	0.002*£
Glucose levels	0.99	0.98 1.01	0.404	0.99	0.98–1.00	0.195¥
Maternal age	1.02	0.94–1.10	0.615	0.95	0.86–1.06	0.366
Pre-pregnancy Maternal BMI	1.13	1.04–1.23	0.004
Maternal BMI at delivery	1.21	1.11–1.33	< 0.001*	1.22	1.10–1.35	< 0.001*
Parity	1.56	1.15–2.13	0.004	1.37	0.94–1.99	0.103
Gestational age at delivery (weeks)	1.31	0.95–1.79	0.098	1.53	1.02–2.30	0.039
Ethnic group
Others	1.00	Reference	Reference	1.00	Reference	Reference
Yoruba	0.37	0.08–1.81	0.220	0.73	0.12–4.37	0.728
Igbo	0.38	0.08–1.90	0.239	0.47	0.08–2.70	0.398
Fetal sex
Female	1.00	Reference	Reference	1.00	Reference	Reference
Male	3.70	1.77–7.72	< 0.001*	4.4804	1.85–10.82	0.001*

£: The p-value was obtained from a different (second) model.

¥: The p-value was obtained from the third model.

* Statistically significant at P-value < 0.001.

Variables in the multivariable model I includes Insulin, maternal age, maternal BMI at birth, parity, gestational age, ethic group, and fetal sex.

For models II and Model III, insulin was replaced with IGF-1 and glucose respectively

Spearman’s rank correlation of cord blood IGF-1 with neonatal anthropometry indicates positive correlation with birth weight (r = 0.47, P-value < 0.001), fetal length (r = 0.30, P-value = 0.0002) and OFC (r = 0.37, P-value < 0.001) (Fig 2). Similarly, cord blood Insulin had statistically significant positive correlation with fetal length (r = 0.19, P-value = 0.0201) and birth weight (r = 0.18, P-value = 0.0270) but there was no statistically significant relationship with OFC (r = 0.13; P-value = 0.1157). The relationship between glucose and the birth anthropometry, showed negative correlation and was not statistically significant for the Fetal length (r = -0.002; P-value = 0.98), OFC (r = -0.114; P-value = 0.16) and birth weight (r = -0.061; P-value = 0.46).

Fig 2

Spearman’s correlation coefficient of cord blood insulin and IGF-1 with birth weight.

Discussion

The study objectives were to measure fetal cord levels of IGF-1 and insulin and assess their correlation with the anthropometric indices of the neonates. Compared to normosomic neonates, insulin and IGF-1 were significantly higher in macrosomic neonates. Macrosomic neonates had median insulin and IGF-1 of 4.84IU/ml and 85.09 ng/ml, respectively. Maternal BMI at birth, fetal gender, and fetal cord serum IGF-1 were the significant predictors of fetal macrosomia on multivariable regression. Also, there was a weak positive correlation of cord blood IGF-1 with fetal anthropometric indices of birth weight, fetal length, and OFC. While cord blood Insulin significantly correlated with fetal length and birth weight, it had no statistically significant correlation with OFC.

Fetal growth regulation differs significantly from the postnatal growth process. While maternal and placenta factors, especially in the late trimester, influence fetal growth, childhood growth is exclusively influenced by genetic factors. This study reported a statistically significant association of fetal macrosomia with maternal parity, maternal height, previous history of big baby, and gender of the baby, with male dominance in macrosomia. Aside from maternal characteristics reported to influence birth weight, the neonates’ gender has also been associated with macrosomia in previous studies [24-26].

Several authors have reported the role of insulin, IGF-1, and glucose in the determination of birth size, and by extension, fetal macrosomia in addition to fetal anthropometry [19, 20, 22]. Unlike the link of maternal hyperinsulinemia to excessive fetal growth as proposed by Pedersen’s theory [27], most women in this study were euglycemic, including few diabetic women with good blood sugar control. Hence, maternal hyperglycemia with fetal hyperinsulinemia has little contribution to fetal macrosomia in this study. While Jacksic et al. reported the lower cord blood glucose levels in macrosomic babies due to higher insulin [26], Wizniter reported IGF-1 as the primary driver for growth because of higher insulin levels in macrosomic neonates [28]. However, the conclusions of Wizniter et al. were limited by the small sample size of the macrosomic study population [28]. In a sample of euglycemic mothers, insulin and IGF-1 levels were significantly higher in macrosomic neonates, with IGF-1 levels strongly associated with birth weight [26, 28, 29].

In this study, the level of IGF-1 was significantly higher in macrosomia compared to controls (85.09 vs. 53.94: p-value = 0.0001). A previous study had reported a strong positive linear relationship between cord IGF-1 birth weight and birth length [19]. Despite these reports, Chiesa et al. reported no significant difference in insulin levels of macrosomic and normosomic neonates [21], with authors attributing their findings to IGF-1 level’s regulation by insulin. However, the study involved only 27 macrosomic babies [21].

This study reports a correlation of fetal length, head circumference, and birth weight with Insulin and IGF-1. Another study reported a correlation (r = 0.39 p<0.0001) between insulin levels and birth weight [30]. Evaluating the implications of malaria parasitemia on fetal anthropometric measures in a sample of Nigerian neonates, Ayoola et al. reported a significantly strong correlation of birth weight, insulin and IGF-1 [22]. However, there was no significant correlation between birth length, OFC, and Insulin among their cohort of babies. Similarly, their study did not reveal any correlation between birth length, OFC, and IGF-1. This difference between our results and those of Ayoola et al. might be due to anthropometric measurements of neonates whose mothers had malaria parasitemia [22].

This study provides the scientific basis for a relatively common clinical condition of fetal macrosomia. In Nigeria, fetal macrosomia incidence has been reported to be 5.5%- 8.1% [24, 31, 32]. It provides an association of IGF-1 and Insulin with fetal macrosomia in a largely non-diabetic population of pregnant women. The understanding that high IGF-1 and Insulin concentrations in fetuses are predispositions to fetal macrosomia might open a new vista of prenatal assessment of fetal IGF-1 and insulin as well as interventions to prevent fetal macrosomia and its associated maternal and fetal morbidities like higher caesarean section rates, operative vaginal deliveries, increased duration of birth, shoulder dystocia and genital lacerations [24, 31, 32].

Our study is the first in Nigeria to prospectively evaluate the relationship between IGF-1 and fetal macrosomia in euglycaemic mothers, thereby contributing to the growing evidence of the role of IGF-1 in growth and metabolic disorder in our environment. A strength of the study is the recruitment ration of 2:1 for cases and controls, unlike the usual 1:1 recruitment ratio. The collection of blood sample at birth, and fetal anthropometric measure within 24 hours of birth supports the results. We report a significant association between insulin and IGF-1 as a macrosomia marker, with glucose contributing little to fetal macrosomia, contrary to Pedersen’s theory. The study limitations may include the recruitment of only euglycemic mothers and the use of Hospital controls. Yet, this research’s findings are like the reports of macrosomia in diabetic women. Due to the equivocation of the roles of hyperinsulinemia in fetal growth, there is the need to evaluate other fetal growth biologic fuel, with the hope of identifying the stimulus for the hyperinsulinaemic states of macrosomic babies.

Conclusion

Fetal insulin and IGF-1 were significantly higher among macrosomic neonates compared to normosonic neonate. Among participating mother-neonate dyad, maternal BMI at birth, neonate’s gender, and fetal cord serum IGF-1, and serum insulin were significantly associated with fetal macrosomia.

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27 Oct 2021

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Reviewer #1: General comments

It is difficult to understand at the time of reading what the rationale of the study is and why it seems so important in the Nigerian population to know if there is an association between fetal insulin and IGF1 levels and macrosomia. What clinical impact do the authors expect from their findings, if any?

Moreover, there is a discrepancy between the announced objective (relationship between fetal insulin, IGF1 and macrosomia) and the results and the conclusion which essentially concerns the maternal, neonatal and biological clinical predictive factors of macrosomia. Why is the study and the collected samples from 2015 and only submitted for publication in 2021?

Specific comments

MM

Justify the choice of a 2/1 control case study?

Why did you not match the controls on BMI and parity, which are major confounding factors?

what is the rationale for the evaluation of neonatal anthropometric measurements of the choice of occipitofrontal circumference ? It does not seem that it is a representation of the neonatal fat mass. What is Digital weighing scale ?

The definition of macrosomia is not given and if it is a question of babies weighing more than 4000g as it seems to appear in the results, then some babies are not macrosome since this depends on the gestational age at birth and therefore on the weight curves at birth. Indeed at term 4000kg is not a fetus > 90th percentile and is not considered as macrosomic newborn. Please give the definition of macrosomia and justify this choice for the interest of the study.

Table 1 should be simplified and fit on one page. On the other hand, as indicated, the groups between macrocosmic and non-macrocosmic fetuses are not comparable and BMI and parity appear as confounding factors and limit the interpretation of the results.

Results

Line 204 It is not indicated either in MM or in the results which confounding factors were entered in the logistic regression model. Please indicate these details

When interpreting the results in Table 2, the sentence maternal BMI at birth (P205

value =0.003), fetal gender (P-value < 0.001), fetal cord serum PGF-1 (< 0.001), and insulin

206 assay (P-value =0.027) were the statistically significant predictors of fetal macrosomia should be changed to "were significantly associated of ..." This is not a prediction model

Similarly, in the conclusion, the word prediction should be changed to association.

Discussion

It seems when reading the discussion that it has been reported in non-diabetic macrocosmic fetuses an increase in insulin and IGF1 and this study just reports the same results but in a Nigerian population. I have difficulty understanding the clinical impact of these results and this is not at all discussed. Metabolic consequences in the macrosomic newborn? Particular follow-up of these children?

There are many points to review in the objective, methodology and results in order to better understand the message that the authors wish to give in this article

**********

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Reviewer #1: No

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10 Dec 2021

6th December 2021

The Editorial Board

PlosOne

Thank you for a timely review of the manuscript. The authors have reviewed the comments/ edits of the reviewers and have made changes as suggested by them. Where it was impossible to make the suggested review, we have provided reasons for our position. For ease of identification, we have provided files with tracked changes and clean copy. Kindly find below a point-by-point response to the reviewers’ comments.

When submitting your revision, we need you to address these additional requirements.

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2. Please amend your current ethics statement to address the following concerns: Please explain why written consent was not obtained, how you recorded/documented participant consent, and if the ethics committees/IRBs approved this consent procedure.

Response: We obtained ethical approval for written consent, which was the consent procedure for this research. We have now indicated we obtained written consent in the manuscript as: “Informed written consent was obtained from the mothers. The ethics committee approved the consent and all other aspects of the research. There was however no potential harm to the mothers and babies as this was an observational study with no intervention. Autonomy and confidentiality was maintained throughout the study” line 169 – 173 (page 7)

Kindly note that informed written consent was obtained from the participants and was originally stated in the research dissertation at page 34 as published by the National Postgraduate Medical College of Nigeria. 1089-Article Text-6613-1-10-20190415 (3).pdf

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

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We will update your Data Availability statement to reflect the information you provide in your cover letter.

Response: We have uploaded the data supporting our manuscript as supplementary file named S1.

3. Please amend the manuscript submission data (via Edit Submission) to include author Oluwakayode O Akinmola.

Response: This has been done. The correct name of Olukayode Akinmola has been added.

4. Please amend your authorship list in your manuscript file to include author Kayode Akinmola.

Response: This has been done. The correct name of Olukayode Akinmola has been added

5. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 2 in your text; if accepted, production will need this reference to link the reader to the Table.

Response: This has now been done. Kindly refer to line 240, page 16.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: No

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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Reviewer #1: No

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Reviewer #1: No

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General comments

It is difficult to understand at the time of reading what the rationale of the study is and why it seems so important in the Nigerian population to know if there is an association between fetal insulin and IGF1 levels and macrosomia.

Response: We have recast the manuscript and provided further study justification. We stated that the two main pathways of influence of fetal weight was fetoplacental and genetic factors. One of the feto-placental factors is the elaboration of insulin and IGF-1. In most paragraphs of the introduction section, the study justification was presented. Specifically, and in line with the reviewer’s comment, we stated:

“Establishing an association between IGF-1, insulin and macrosomia among Nigerian babies can aid further interventional research on the feto-maternal pathophysiology of macrosomia and its attendant immediate maternal complications at birth and later life associated disease risks reductions” line 123- 126 (Page 5)

What clinical impact do the authors expect from their findings, if any?

Moreover, there is a discrepancy between the announced objective (relationship between fetal insulin, IGF1 and macrosomia) and the results and the conclusion which essentially concerns the maternal, neonatal and biological clinical predictive factors of macrosomia.

Response: The authors did not see any discrepancies between the research objectives, results and conclusion. We reported on our primary objective of the association between IGF-1 and macrosomia. We further conducted multivariable regression modelling to assess the association between IGF-1 and macrosomia. We then reported other secondary findings.

Why is the study and the collected samples from 2015 and only submitted for publication in 2021?

Response: the observed interval between research and publication is correct. We believe in transparent scientific reporting, which was why the dates are as reported. Essentially, the study was conducted in part fulfilment for the requirement of the award of the Fellowship of the National Postgraduate Medical College of Nigeria. It is being reported in a scientific journal now because the candidate’s mentors have encouraged him to disseminate his research findings in a peer-reviewed journal.

Specific comments

MM

Justify the choice of a 2/1 control case study?

Response: Case control studies might have case: control ratio of 1:1, 1:2, 1:3, 1:4 or vice versa. We adopted a 2:1 ratio by recruiting 100 cases and 50 control to minimize the challenges of enrolment of controls, and to reduce the study duration and costs. Furthermore, some sub-analyses were conducted among the cases. Thus, a larger sample of the cases will improve the conclusion of such intraclass analyses.

Why did you not match the controls on BMI and parity, which are major confounding factors?

Response: Known confounders in research may be handled during participants recruitment and data analysis. Due to the difficulty of matching using more than one variable, matching was done with only one variable. However, we utilized multivariable regression modelling to correct for other known confounding variables, including parity. We have now added parity as a variable in the multivariable model (Table 2)

what is the rationale for the evaluation of neonatal anthropometric measurements of the choice of occipitofrontal circumference ? It does not seem that it is a representation of the neonatal fat mass.

Response: We thank you for this question. We decided to show all the anthropometric measurements, including the occipitofrontal circumference. Although, at the moment OFC does not have biologic direct correlation with the neonatal fat mass, we felt that showing all the parameters might be valuable to the literature in future. If the reviewers and indeed the editor strongly feel otherwise, the authors will remove OFC as an anthropometric measure.

What is Digital weighing scale ?

Response: The weighing scale used for participants weight measurement has been further described. Line 147 - 148

The definition of macrosomia is not given and if it is a question of babies weighing more than 4000g as it seems to appear in the results, then some babies are not macrosome since this depends on the gestational age at birth and therefore on the weight curves at birth. Indeed at term 4000kg is not a fetus > 90th percentile and is not considered as macrosomic newborn. Please give the definition of macrosomia and justify this choice for the interest of the study.

Response: For this study, the definition of macrosomia is birth weight weight ≥ 4000g. This has been defined in the introduction (line 102 - 102) and methodology (line 138) with references. Also, we used an absolute weight of 4000g at birth as it is more pragmatic and will avoid the use of different cut-off weights for different neonates.

Table 1 should be simplified and fit on one page.

Response: The Table has been re-designed to fit 2-pages in Landscape format as it was not possible to make it a page.

On the other hand, as indicated, the groups between macrocosmic and non-macrocosmic fetuses are not comparable and BMI and parity appear as confounding factors and limit the interpretation of the results.

Response: Table 1 is a bivariate analysis. We corrected for the confounding variables in Table 2. Table 2 showed the univariable and multivariable analysis that corrected for BMI and parity. We re-analyzed the multivariable model by including parity in the model. This did not change the point estimates or direction of association of the relationship between insulin and the outcome.

Results

Line 204 It is not indicated either in MM or in the results which confounding factors were entered in the logistic regression model. Please indicate these details

Response: Thank you for this observation. This has now been stated in the methods (line 192 - 194) and as legend of Table 2

When interpreting the results in Table 2, the sentence maternal BMI at birth (P205

value =0.003), fetal gender (P-value < 0.001), fetal cord serum PGF-1 (< 0.001), and insulin

206 assay (P-value =0.027) were the statistically significant predictors of fetal macrosomia should be changed to "were significantly associated of ..." This is not a prediction model

Response: This has been corrected (Line 237 -238)

Similarly, in the conclusion, the word prediction should be changed to association.

Response: This has been corrected in the conclusion of the abstract and the conclusion after discussion (Line 331 -332)

Discussion

It seems when reading the discussion that it has been reported in non-diabetic macrocosmic fetuses an increase in insulin and IGF1 and this study just reports the same results but in a Nigerian population. I have difficulty understanding the clinical impact of these results and this is not at all discussed. Metabolic consequences in the macrosomic newborn? Particular follow-up of these children?

There are many points to review in the objective, methodology and results in order to better understand the message that the authors wish to give in this article

Response: This is a basic science research that provides the biochemical basis for fetal macrosomia, a relative common pregnancy complication in Nigeria. On Page 20, lines 308 - 315 of the revised manuscript, the authors have included clinical implications of the study findings, including a potential use of prenatal assay of fetal IGF-1 and insulin to predict fetuses who might develop fetal macrosomia.

6. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Letter of rebuttal_Final.doc

Click here for additional data file.

24 Jan 2022

17 Mar 2022

Department of Obstetrics and Gynaecology,

College of Medicine,

University of Lagos,

Lagos, Nigeria.

16th March, 2022

The Editorial in Chief

PlosOne

Dear Editor-in-Chief,

Revised manuscript Titled Fetal macrosomia, fetal Insulin, and Insulin-like growth factor- 1 among neonates in Lagos, Nigeria: A case-control study

We are grateful for the favorable outcome of our above titled manuscript. We have further edited the manuscript in line with the recommendations of the editor. Kindly find below a point-by-point response to the reviewers’ comments. We have uploaded a clean copy and a manuscript with tracked changes.

Editor’s comment:

The aim of the present manuscript by Akinmola et al was to measure fetal insulin and insulin-like growth factor-1 (IGF-1) levels in macrosomic fetuses delivered at term in Lagos State, Nigeria. Overall, the research was aimed at determining potential correlations between both hormones with fetal anthropometric variables. Authors provide evidence that maternal BMI at birth, neonate’s gender, and fetal cord serum IGF-1 and insulin were correlated with fetal macrosomia. Authors emphasize that this association might help identify these hormones as important predictive factors to prevent macrosomia. Hence, the paper might bear important clinical relevance.

The present manuscript constitutes a revised version of the paper. Authors have addressed reviewer’s concerns in a satisfactory manner. In particular, authors expand on the rationale of the study and better elaborate the justification for the study. In addition, authors clarify a number of methodological issues in the revised version.

In summary, the present manuscript is a concise and straightforward analysis of the link between neonate insulin, IGF-1 and macrosomia. While mainly of a descriptive nature, the paper includes valuable informmation.

Authors’ response: We are grateful for the favourable and fair assessment of our manuscript

Editor’s comment:

Minor points:

Lines 50, 203, 207, 238, 300, : should be IGF-I, not PGF-1.

Authors’ response:

We have changed PGF-1 to IGF-1 throughout the manuscript. We further utilized the “find” in Microsoft word to ensure all PGF-1 has been changed to IGF-1.

Once again, we are grateful for the opportunity to publish in your prestigious journal

Yours faithfully,

Dr Babasola Okusanya (on behalf of co-authors)

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

21 Mar 2022

Fetal macrosomia, fetal Insulin, and Insulin-like growth factor- 1 among neonates in Lagos, Nigeria: A case-control study

PONE-D-21-16686R2

Dear Dr. Okusanya,

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PLOS ONE

Additional Editor Comments (optional):

Authors have satisfactorily addressed reviewer's comments.

Reviewers' comments:

18 Apr 2022

PONE-D-21-16686R2

Fetal macrosomia, fetal Insulin, and Insulin-like growth factor- 1 among neonates in Lagos, Nigeria: A case-control study

Dear Dr. Okusanya:

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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