In this project three studies examined two possible explanations and one possible preventive intervention to early cessation of exclusively breastfeeding in premature infants.

Study 1 The content of protein in the milk of mothers, who delivers prematurely, is about a third higher than in the milk from the mother who delivers on time. The nutritional composition changes over time and the content of protein decrease. Therefore the premature infant is at risk of protein deficiency. While the infant is feeding by tube this decreasing content of protein can made up by adding, while it is more difficult when the infant is exclusively breastfeeding. The hypothesis is that reduced protein content in breast milk is associated to a fewer number of days where the premature infant is exclusively breastfed.

Study 2 The premature infant is characterized with immature muscle with a low tension and therefore, a low ability to eat its needs by breastfeeding the first period. The transfer of milk from mother to child is an interaction between the mothers and her milk ejection reflex that establish a positive pressure on the milk and the child that have to establish a vacuum. The hypothesis is that the premature infants suction power is too weak to establish sufficient intraoral vacuum to ensure milk transfer from the breast to the infant and it can be related to a fewer number of days where the infant is exclusively breastfed.

Study 3 The premature infants low muscle tone and its immaturity also influence on the organization and the quality of movements, marked as neuro motor processes. These processes form the oral motor base supporting movement which involves the infant ability to establish vacuum. The hypothesis is that Oral Stimulation for a specific program in 5 minutes before the minimum 2 meals per. day for at least 14 days increases the preterm infant's ability to create intra oral vacuum and thus the power to transfer milk from the breast, thereby extending the number of days when the infant is exclusively breastfed.

200 infants are included consecutively, as a recurring cohort in all 3 studies. In Study 1 the mothers' milk is analyzed in order to the content of protein. In Study 2 the infant suction is assessed by vacuum measurement. In study 3 the families are randomized to an intervention or control group and parents off 100 infants are guided by occupational therapists in a program of oral stimulation of their child.

The purpose of this project, which consists of three studies, is to investigate whether insufficient protein in breastmilk and / or inadequate intra oral vacuum of the infant can explain early breastfeeding cessation in the premature infant. Furthermore to test whether the breastfeeding period may be extended by oral stimulation.

The rate of breastfeeding in mothers of infants born prematurely is lower than in mothers who give birth on time. A Danish survey shows that about 70% of premature are on exclusive breastfeeding at discharge and that this rate is decreasing to about 30% six weeks after discharge. Breastmilk is the healthiest nutrition for the newborn and based on WHO's strategy for the feeding of infants Board of Health recommends that newborn infants, including premature, as far as possible are exclusively breastfed for six months. Exclusively breastfeeding means that the infant only gets the breastmilk of the mother. Exclusively breastfeeding has health implications for both mother and infant. Breastfeeding reduces lactation incidence of infectious diseases, improves cognitive development and visual function in the infant and reduces the risk of developing later obesity. In addition, the level of immune protective factors and anti-inflammatory elements is high in the mother's milk to the preterm infant and therefore reduce the infant's increased risk of early and severe infections, and death late effects. Furthermore, breastfeeding may reduce the risk of premature infant's abnormal vessel growth in the eye, and appears to have a protective effect on the disease being of significant excess mortality primarily attack the intestines of the preterm infant (necrotizing enterocolitis).

Problem Area / factors in milk composition with the importance of breastfeeding Breastmilk contains the necessary nutritional components and energy that the infant needs. Therefore, the milk of the mother delivering early have a higher content of fat and protein, and thereby a higher level of energy comparing with the milk from the mother who have given birth to term. The content of protein in the milk of the mother who gives birth prematurely is about 12 grams per liter and thus approximately one-third higher than that from the mother who gives birth on time. The nutritional composition of breastmilk vary considerably over time and the content of protein is decreasing why the premature infant are at risk of protein deficiency and thereby insufficient growth. During the hospital stay, and while the infant are feed by tube this decreasing protein content are made up by adding, while it is more difficult when the infant breastfeed exclusively. Protein is important for the infant's growth and strength and a local statement from Hvidovre Hospital found that 18% of all hospitalized preterm infants in 2014 were added additional protein to breastmilk, because growth was not sufficient. The question is whether reduced protein content in breastmilk has a correlation with the number of days where preterm infants are exclusively breastfed?

Problem Area / intra oral vacuum strength of the infant with significance for breastfeeding The period from birth to the premature infant feed exclusively at the breast without supplementary tube feeding varies from weeks to months, depending on how early the infant is born. The premature infant is characterized as being immature and with low muscle tone and therefore, a decreased ability to breastfeed the first time. The transfer of milk from mother to infant is an interaction between the mother and her milk ejection reflex creates a positive pressure on the milk and the infant creates a vacuum. If the infant not create a vacuum at the back of the mouth, the nipple of the mother is just pushed together and the infant is not transferred milk from the breast to the oral cavity. The amount of milk is regulated in relation to demand and a weak or shortage of vacuum will cause a shortage of milk transfer, which in turn physiologically will lead to less milk production and thereby decreasing amount. Studies that investigates this process and measured intra oral vacuum, shows all the importance of the infant's contribution by milk transfer, but does not answer to the extent the premature infants ability to create vacuum importance of breastfeeding establishment and maintaining. The question is whether the premature infants sucking strength is too weak to establish sufficient intraoral vacuum to ensure milk transfer from the breast to the infant and it can be related to a fewer number of days where the infant is exclusively breastfed.

In addition to the premature infants muscle tone has its immaturity also affect the organization and quality of body movements. Organization and quality in the movements may be referred to neuro motor processes and form among others the oral motor base supporting the movements involving the infant's ability to create vacuum. The processes preceding the creation of vacuum and studies have shown that by early intervention with oral stimulation in the form of stimulation of the nerves and muscles in and around the mouth, it can be oral motor basis strengthened. Studies where the infant carefully is stimulated with a finger, showing that the premature infant's muscles and sucking reflex can be influenced so that it is able to suck a larger volume in a shorter time in the bottle compared to the infant that is not stimulated orally. This presents an opportunity to influence the premature infant´s strength and maybe increase the ability to create intra oral vacuum affected by oral stimulation as a skill the infant preserves. The question is whether oral stimulation can increase the premature infant's ability to create vacuum and thus increase the power to transfer milk from the breast and thereby extending the number of days when with exclusively breastfed? The 3 studies are based on the same cohort. The cohort is identified and recruited at the Neonatal Intensive Care Unit at Hvidovre Hospital and relevant families where the mother delivers prematurely are included consecutively in accordance to the inclusion and exclusion criteria. The cohort will be demographically representative of everyday life in clinical practice with various family formation, education levels and socio-economic background conditions.

Power calculations are based primary outcome: exclusively breastfeeding duration and made to show effect on exclusively breastfeeding duration between the intervention- and the control group. Requirements for power calculation are a mean value of the vacuum set at 100% with a standard deviation (SD) of 26%. To detect the smallest clinically relevant difference of 13%, equivalent to half a SD and with an expected drop out of 20%, which included 200 infants (100 infants in each group) in the study (alpha = 0.05 and beta = 0.1 ). There hospitalized annually on average 300 preterm infants in the neonatal section. It is expected about 100 infants to meet the criteria for inclusion, thus achieved at approximately 2 year.

By inclusion the family is anonymized by allocating an ID number. When multiple assigned each infant this ID number and a one of letters a, b or c. All data and all measurements is obtained / made and recorded as far as possible of the project manager or alternatively the project nurse.

6 weeks after the date for the expected terminator date either the family are visiting the Neonatal section or the project manager/project nurse visiting the family in order to a status of breastfeeding, vacuum measurement and the infant's growth. In addition the family is contact by telephone the week in which the infant's postnatal age is 6 months. Here is recorded breastfeeding status, the time of any termination of exclusive breastfeeding and the infant's weight at GP visit 5 month old. The infant's growth is defined by its naked weight, Length and head circumference. Previous studies show a number of factors which affect breastfeeding duration. Features concerning socio-demographic background variables, perinatal and psychosocial conditions will gathered by the project manager or project nurse using a questionnaire in an interview with the family and form data in the analyzes. Participation in the project is safe and not associated with risk or discomfort for either mother or infant.

STUDY 1 / PROTEIN IN BREASTMILK Data are obtained in the human milk section at the hospital. Volume is obtained in milliliter (ml.) and protein content in gram pr. 100 ml. by using Miris Human Milk Analyzer according to the usual practice. Miri is the only European equipment that analyzes human milk. The test sample is 10 milliliters and the result given within a minute and logged into the machine and then transferred to the project database. When the mother is exclusively breastfeeding, we ask for 10 ml. Manual hand compressed breast milk. The analyzed milk can´t be reused and is therefore destroyed.

STUDY 2 / WEEK STRENGTH ASSESSMENT BY VACUUM MEASUREMENT Sucking strength of the premature infant is defined as the ability to create intra oral vacuum during sucking.

The intra oral vacuum of the premature infant is assessed by measuring the vacuum in the unit of mBar and are primary made by the project manager which is blinded to know if the infant has received oral stimulation or not. The measurement is made when the infant is awake and show readiness to suck. For vacuum measurement is a manometer selected with a bottle pacifier, which fits a Calmaflaske made by the firm Medela. The pacifier is shaped and is similar in function a breast and extra small so that it matches the premature mouth of a premature infant. As in a completely closed system the sucking of the infant is registered on this connected manometer. In order to obtain a measure of reproducibility of the method all vacuum measurements is carried out at 3 consecutive measurements. The advantage of the 3 measurements within a short time is to determine the intra-individual variability, but only if the infant is motivated and show redlines for measurement 3 times. The equipment is developed in cooperation with the Technical University of Denmark.

STUDY 3 / randomized INTERVENTION STUDY / ORAL STIMULATION Infants whose mothers is included in the cohort is randomized to receive / not receive oral stimulation when the infant's postnatal age is at least 32 + 0 weeks, as it is the time when the infant is able to coordinate sucking and swallowing reflex. In multiple birth all infants is randomized to the same group. The list is generated block randomized in blocks of 10

Families were randomized to the intervention group granted in total one hour instructions in a program of oral stimulation by one of two occupational therapists with experience in oral stimulation of preterm infants. The program is in collaboration with the project team developed and described by the Department of Physiology and Occupational therapy at Hvidovre Hospital. The first guidance for parents has duration of approximately half an hour and then follow-up once or twice, depending on the needs of the family. To maintain consistent intervention over time is the program of oral stimulation written in a script, where the family also must record the following variables: date and time of oral stimulation. In occupational therapy the guidance and in the script examines the signs that the infant exhibits when it is ready / not ready for stimulation. The family also sees a movie where these signs and the program of oral stimulation review auditory and visual. Families in the control group is not seeing the movie, do not receive the script or the supervision of occupational therapists and these infants do not get oral stimulation. Both groups receive instructions after usual practice i.e. guidance and elements that promote breastfeeding example, skin to skin contact and compression.

Inclusion Criteria:

• mothers: with premature infant i.e. the infant's gestational age is equal to or maximum of 36 + 6
• intend to breastfeed.
• The mother or father / other primary caregiver understand Danish, Norwegian, Swedish or English.

Exclusion Criteria:

• mothers: will not / can´t breastfeed because of illness or abuse such as: HIV and drugs.
• In addition, if the infant has a disability which makes it difficult to breastfeeding or if the infant is physiologically unstable, for example due to reduced lung function.