European Journal of Cardiovascular Medicine

Exclusive breast feeding is established as the standard of care. However, lactation failure is a significant hindrance to meet such care in a sizeable population. Delayed initiation of lactation is defined as non-production of milk within 72 hours of childbirth [1]. Non-initiation of lactation is defined as non-production of milk after childbirth. Recent study conducted by Mukunya et al and WHO statement projects approximately 48.2 % incidence of delayed lactation (DL) on a global level [2,3]

Abnormalities of nipple, oral cavity anomalies of newborn, faulty technique, mother-baby-separation and early introduction of artificial feeds are important factors. Apart from these causes, many mothers show failure of initiation of lactation due to possible hormonal dysregulation [4]. Obesity, Diabetes mellitus, dyslipidemia, hypertension and hypothyroidism may act as important factors [5]. The root cause may be linked to insulin resistance (IR).

Insulin resistance is a condition in which the end organs are non-responsive to insulin stimulation and glucose metabolism. This initially leads to overproduction of insulin by beta-cells of pancreas resulting in a state of hyperinsulinemia [6]. This finally precipitates into diabetes mellitus. IR (pre-diabetic state) is also looked upon as a state of metabolic syndrome. Initially Metabolic syndrome

was identified by the presence of 3 or more diagnostic points. However, the American Association of Clinical Endocrinology definition of 2016 is more adaptable for clinical practice. American Diabetes Association in 2016 further added glycosylated hemoglobin, HbA1c as an important factor [7].

Thus, IR is hypothesized to be a key factor in non- production of milk after childbirth.

To estimate the association between hormonal factors and factors of insulin resistance with delayed initiation/non- initiation of lactation.

1. Study design: Prospective observational study
2. 124 mothers, Gr-A 62 mothers with delayed/non-lactation (DL), Gr-B 62 patients with normal-lactation (NmL) attending Lactation Clinic were
3. Study is planned with inclusion, exclusion criteria and withdrawal criteria

Inclusion criteria are the mothers with delayed lactation (lactation established after 72 hours following delivery) or non-lactation (lactation not established)

Exclusion criteria are anatomical abnormalities of mother’s breast and NAC, anatomical abnormalities of the newborn which include oral and oropharyngeal abnormalities, faulty feeding and latching techniques, prematurity or baby admitted in NICU after delivery, mother very sick after delivery, or there is separation between mother and baby following birth.

Withdrawal criteria include mother did not want to participate in the study or lost in follow up.

Subjects attending the out-patient-department, lactation clinic or admitted in Institute of child health are eligible to enrol in this study.

Sampling method: Simple Random Sampling is used. Eligible patients (Gr-A) with the inclusion criteria are enrolled along with patients with normal lactation (Gr-B) the age group being equally matched, 18 – 40 years. Inform consent is taken and the form is signed by the mothers.

In both the groups, mother’s history of onset of lactation, i.e. colostrum is noted along with history of hypertension, diabetes mellitus and hypothyroidism. Mother’s history of PCOS, infertility is also noted down. Feeding and latching techniques and milk flow is examined. On clinical examination of mother, breast and NAC abnormalities are checked and on clinical examination of the newborn anatomical abnormalities of oral cavity and oropharynx is also checked. Clinically BMI, waistline, waist-hip ratio, systolic and diastolic blood pressure of the mothers are examined in both the Groups A & B.

To check biochemical parameters, fasting blood samples are drawn from the mothers of both the groups to check lipid profile (serum cholesterol, triglycerides, HDL, VLDL, LDL), serum prolactin; serum c-peptide; serum HbA1c, and fasting blood glucose. Post prandial blood glucose is also checked two hours after eating meal.

Amount of blood collected in different vials are: 2 ml of fasting blood collected in EDTA vial for HbA1c, 2 ml of clotted blood in fasting state collected in 2 vials, total volume 4ml for serum c-peptide, serum prolactin, serum lipid profile and thyroid profile. 2ml of fasting blood collected in sodium fluoride vial for fasting blood sugar and 2ml of blood 2hours following meal collected in sodium fluoride vial for postprandial blood sugar.

Data were entered into a Microsoft excel spreadsheet and then analysed by SPSS (version 27.0; SPSS Inc., Chicago, IL, USA). Without other qualification, 'chi-squared test' often is used as short for Pearson's chi-squared test. Unpaired proportions were compared by Chi-square test or Fischer’s exact test, as appropriate.

Once a t value is determined, a p-value can be found using a table of values from Student's t-distribution. If the calculated p-value is below the threshold chosen for statistical significance (usually the 0.10, the 0.05, or 0.01 level), then the null hypothesis is rejected in favour of the alternative hypothesis.

P-value ≤ 0.05 was considered for statistically significant.

Table: Distribution of mean All parameters: Group

Age versus delayed lactation /Non lactation: In DL Group, the mean Age (mean± s.d.) of patients was 27.7258± 5.0705. In NmL Group, the mean Age (mean± s.d.) of patients was 27.1290± 4. 7855.However, the distribution of mean Age with Group was not statistically significant (p=0.5016). (Table 1)

BMI versus Delayed/Non lactation: In DL Group, the mean BMI (mean± s.d.) of patients was 29.1597± 4.8575. In NmL Group, the mean BMI (mean± s.d.) of patients was 23.9411± 3. 4888.Distribution of mean BMI with Group was statistically significant (p<0.0001) in DL group. (Table 2)

Waistline versus Delayed/Non-Lactation: In DL Group, the mean Waistline (mean± s.d.) of patients was 35.2798± 4.1997. In NmL Group, the mean Waistline (mean± s.d.) of patients was 30.9750± 3. 4854.Distribution of mean Waistline with Group was statistically significant (p<0.0001). Mothers with DL had higher waistline circumference. (Table 3)

Blood pressure versus Delayed/Non-Lactation: In DL Group, the mean SBP (mean± s.d.) of patients was 115.6935± 15.5222. In NmL Group, the mean SBP (mean± s.d.) of patients was 109.3387± 10.6586. Distribution of mean SBP with Group was statistically significant (p=0.0089). (Table 4)

In DL Group, the mean DBP (mean± s.d.) of patients was 76.9355± 11. 8209.In NmL Group, the mean DBP (mean± s.d.) of patients was 71.2258± 8. 6866. Distribution of mean DBP with Group was statistically significant (p=0.0027). (Table 5)

Both systolic and diastolic blood pressure were significantly higher in DL group compared to NL group.

Blood glucose level in DL vs NmL group: The mean Fasting blood glucose (mean± s.d.) of the mothers in DL was 87.9194± 14.1181vs the mean FBS (mean± s.d.) in NmL group of patients was 83.4306± 8. 9333. Distribution of mean FBS with DL Group was statistically significantly higher (p=0.0364). (Table 6)

In DL Group, the mean PPBS (mean± s.d.) of patients was 111.9355± 24. 9582.In NmL Group, the mean PPBS (mean± s.d.) of patients was 101.9677± 10.6816. Distribution of mean PPBS with DL Group was statistically significantly higher (p=0.0045). (Table 7)

HbA1c versus Delayed/Non lactation: In DL Group, the mean HbA1c (mean± s.d.) of patients was 5.4795± 9396.In NmL Group, the mean HbA1c (mean± s.d.) of patients was 4.7192± .7939. Distribution of mean HbA1c with DL Group was statistically significantly higher (p<0.0001). (Table8)

C-peptide versus Delayed/Non-lactation: In DL Group, the mean C-peptide (mean± s.d.) of patients was 2.2729± 1.0869. In NmL Group, the mean C-peptide (mean± s.d.) of patients was 2.1444± 1.3173. Distribution of mean C- peptide with Group was not statistically significant (p=0.5545). (Table 9)

Lipid profile versus Delayed/Non-lactation: In DL Group, the mean TG (mean± s.d.) of patients was 117.7258± 42. 7172. In NL Group, the mean TG (mean± s.d.) of patients was 100.5484± 23.5620. Distribution of mean TG with Group was statistically significant (p=0.0064). (Table 10) In DL Group, the mean TC (mean± s.d.) of patients was 179.7097± 33. 0783.In NL Group, the mean TC (mean± s.d.) of patients was 180.0968± 29.4839. Distribution of mean TC with Group was not statistically significant (p=0.9453). (Table 11)

In DL Group, the mean LDL (mean± s.d.) of patients was 112.9194± 28.0522. In NL Group, the mean LDL (mean± s.d.) of patients was 114.1290± 30. 7467.Distribution of mean LDL with Group was not statistically significant (p=0.8194). (Table 12)

In DL Group, the mean HDL (mean± s.d.) of patients was 47.0000± 9.1365. In NL Group, the mean HDL (mean± s.d.) of patients was 46.3710± 12.5740. Distribution of mean HDL with Group was not statistically significant (p=0.7505). (Table 13)

The result analysis shows that serum Triglyceride level is significantly higher in DL group.

Thyroid profile versus Delayed/Non lactation: In DL Group, the mean TSH (mean± s.d.) of patients was 3.1844± 1.9994. In NL Group, the mean TSH (mean± s.d.) of patients was 3.6518± 2.3077. Distribution of mean TSH with both Group was not statistically significant (p=0.2304). (Table 14)

Age and DL :From the data it is observed that , in the delayed lactation and non- lactation group, the percentage of subjects in the age range , 31- 40 years are 29% (18 out of 62 mothers), and in the age range 21-30 years are 67.7% (42 out of 62 mothers).Whereas in the normal lactation group, the percentage of subjects in the age range, 31-40 years are 25.8% (16 out of 62 mothers) and in the age range 21- 30 years are 69.4% (43 out of 62 mothers).

Contrary to the belief that increased maternal age is a hindrance to initiation of lactation, in our study we observed that relationship of maternal age with delayed lactation is statistically insignificant. (p= 0.8481. Nommsen et al has noted that above the age of 30 years there is an association with delayed onset of lactation. In this study the mean age in both the groups is approximately 27 years. This may explain the insignificance of age. This however removes the biasness in the study. [5]

BMI and DL: The results show strong association between higher BMI and DL. BMI is an established marker of insulin resistance (IR). Insulin resistance is a precursor of diabetes. Thus, elevated BMI leads to a state of non- utilization of glucose in the lactogenic cells.

American Association of Clinical Endocrinologists criteria for diagnosis of Insulin resistance (2016) states that Obesity, Serum Triglyceride, HDL (in females), Fasting & Post prandial Blood sugar, Blood pressure and other risk factors (Family history of type2 diabetes mellitus (DM), hypertension (HTN), cardiovascular disease, polycystic ovarian syndrome (PCOS), Sedentary lifestyle, Advancing age, Ethnic groups at higher risk for T2DM, Cardiovascular disease). The study corroborates most features of IR.

Lobular growth of the breast during early pregnancy is promoted by human chorionic gonadotrophin which maintains corpus luteum. It initially secretes progesterone and oestrogen. Following this placental progesterone causes lobular growth and placental oestrogen causes ductal proliferation. Pillay et al described the stages of lactogenesis [8]

Stage 1 lactogenesis: Oestrogen and progesterone initiates lactogenesis from mid-trimester of pregnancy. Prolactin helps in colostrum secretion in the acini, but milk secretion is suppressed under the influence of progesterone. Immunoglobulin is secreted in the colostrum from the alveolar cells converted into lactocytes.

Stage 2 lactogenesis: Following birth of the newborn as the placenta is delivered out, there is decrease in progesterone level. This helps to increase the prolactin level along with cortisol and insulin. All these three hormones stimulate the synthesis of basic components of milk.

Therefore, obesity and insulin resistance are likely lead to reduced lactogenesis.

Stage 3 lactogenesis: As the baby sucks the nipple the nipple-areola complex is stimulated. This provokes oxytocin secretion and milk let down reflex sets in. Myoepithelial cell contraction leads to milk ejection.

Role of Insulin in lactogenesis is fast evolving. Insulin promotes synthesis of lipids, proteins, and carbohydrates. It stimulates the expression of genes leading to in milk protein synthesis [9]. Menzies KK et al concluded that Insulin, Hydrocortisone and Prolactin are the key hormones responsible for milk protein gene expression. However, they could not delineate the exact pathway of Insulin in the whole process. They did mammary explant culture and combine it with lactogenic hormones followed by study of global changes of gene expression using Affimatrix Microarray. They found that 164 genes are responsive to insulin and 18 of them are important in protein synthesis at the level of transcription and post transcription amino acid uptake and metabolism.

Further research in cell lines deciphered that transcription factor Stat5a and Elf5 could be stimulated by insulin even in the absence of prolactin. This indicates the vital role of insulin at molecular biology level [10]

Further analysis of cell lines reveals that transcription factor Stat5a and Elf5, key components of protein synthesis, requires signalling. Stat5a and Elf5 could be induced even in absence of prolactin and in presence of insulin. Prolactin plays an important role in phosphorylating and activating Stat5a, but the expression is only induced by insulin. This established the crucial role of insulin in transcription of milk protein genes.

Insulin influences lactose synthesis and milk fat synthesis by same pathways. [11]

The study has shown significant correlation between the markers of insulin resistance (BMI, Waist circumference, blood pressure, Blood Glucose and HbA1c, Triglyceride level) and delayed /non-lactation across all age group of patients. This corroborates with the above facts that insulin resistance leads to reduced lactogenesis and delayed or non-lactation. The mothers in the DL arm did not have any anatomical abnormalities of nipple. The babies also did not have any orofacial anatomical abnormalities and there was no mother-baby separation. Thus, all possible confounding bias were eliminated from the study.

C-peptide assay was done as a surrogate marker of serum insulin level. Elevated c-peptide may indicate compensatory hyperinsulinemia and reduced c peptide indicated reduced insulin secretion in diabetes mellitus. It was found to be statistically insignificant in this study [12]. However, the correlation with IR is neither very sensitive nor specific. However, insulin assay or HOMA-IR estimation is expected to show more direct corelation.

Hypothyroidism is expected to be an important factor in reduced lactogenesis. In this study, no difference was noted in both the arms. This is because most antenatal check-ups would include hypothyroid screen, and all are medically corrected.

Hypoprolactinemia is a rare entity and is usually associated with Sheehan’s syndrome. This study has also eliminated prolactin as a confounding factor for delayed/non-lactation

Obesity and factors of Insulin resistance directly affects lactogenesis leading to delayed or non-lactation. Mothers with insulin-resistance are prone to lactation failure. To plan public health strategies to achieve “universal breast- feeding goal” as stated by WHO. All mothers must be evaluated in the first trimester of pregnancy and immediately post-partum with special reference to insulin resistance so that prediction of lactation failure can be made, and appropriate therapeutic strategy can be planned. Thus, delayed or non-lactation due to reduced lactogenesis can be looked upon as an effect of lifestyle disorder and metabolic syndrome.

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