Supplementation Of Infant Formula Research Articles

Probiotics in the New Era of Human Milk Oligosaccharides (HMOs): HMO Utilization and Beneficial Effects of Bifidobacterium longum subsp. infantis M-63 on Infant Health.

A healthy gut microbiome is crucial for the immune system and overall development of infants. Bifidobacterium has been known to be a predominant species in the infant gut; however, an emerging concern is the apparent loss of this genus, in particular, Bifidobacterium longum subsp. infantis (B. infantis) in the gut microbiome of infants in industrialized nations, underscoring the importance of restoring this beneficial bacterium. With the growing understanding of the gut microbiome, probiotics, especially infant-type human-residential bifidobacteria (HRB) strains like B. infantis, are gaining prominence for their unique ability to utilize HMOs and positively influence infant health. This article delves into the physiology of a probiotic strain, B. infantis M-63, its symbiotic relationship with HMOs, and its potential in improving gastrointestinal and allergic conditions in infants and children. Moreover, this article critically assesses the role of HMOs and the emerging trend of supplementing infant formulas with the prebiotic HMOs, which serve as fuel for beneficial gut bacteria, thereby emulating the protective effects of breastfeeding. The review highlights the potential of combining B. infantis M-63 with HMOs as a feasible strategy to improve health outcomes in infants and children, acknowledging the complexities and requirements for further research in this area.

Supplemented Infant Formula and Human Breast Milk Show Similar Patterns in Modulating Infant Microbiota Composition and Function In Vitro.

The gut microbiota of healthy breastfed infants is often dominated by bifidobacteria. In an effort to mimic the microbiota of breastfed infants, modern formulas are fortified with bioactive and bifidogenic ingredients. These ingredients promote the optimal health and development of infants as well as the development of the infant microbiota. Here, we used INFOGEST and an in vitro batch fermentation model to investigate the gut health-promoting effects of a commercial infant formula supplemented with a blend containing docosahexaenoic acid (DHA) (20 mg/100 kcal), polydextrose and galactooligosaccharides (PDX/GOS) (4 g/L, 1:1 ratio), milk fat globule membrane (MFGM) (5 g/L), lactoferrin (0.6 g/L), and Bifidobacterium animalis subsp. lactis, BB-12 (BB-12) (106 CFU/g). Using fecal inoculates from three healthy infants, we assessed microbiota changes, the bifidogenic effect, and the short-chain fatty acid (SCFA) production of the supplemented test formula and compared those with data obtained from an unsupplemented base formula and from the breast milk control. Our results show that even after INFOGEST digestion of the formula, the supplemented formula can still maintain its bioactivity and modulate infants' microbiota composition, promote faster bifidobacterial growth, and stimulate production of SCFAs. Thus, it may be concluded that the test formula containing a bioactive blend promotes infant gut microbiota and SCFA profile to something similar, but not identical to those of breastfed infants.

Dietary supplementation of 3'-sialyllactose or 6'-sialyllactose elicits minimal influence on cognitive and brain development in growing pigs.

Sialylated human milk oligosaccharides (HMO), such as 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), are abundant throughout lactation and at much higher concentrations than are present in bovine milk or infant formulas. Previous studies have suggested that sialylated HMO may have neurocognitive benefits in early life. Recent research has focused on infant formula supplementation with key nutrients and bioactives to narrow the developmental gap between formula-fed and breastfed infants. Herein, we investigated the impact of supplemental 3'-SL or 6'-SL on cognitive and brain development at two time-points [postnatal days (PND) 33 and 61]. Two-day-old piglets (N = 75) were randomly assigned to commercial milk replacer ad libitum without or with 3'-SL or 6'-SL (added in a powdered form at a rate of 0.2673% on an as-is weight basis). Cognitive development was assessed via novel object recognition and results were not significant at both time-points (p > 0.05). Magnetic resonance imaging was used to assess structural brain development. Results varied between scan type, diet, and time-point. A main effect of diet was observed for absolute volume of white matter and 9 other regions of interest (ROI), as well as for relative volume of the pons on PND 30 (p < 0.05). Similar effects were observed on PND 58. Diffusion tensor imaging indicated minimal differences on PND 30 (p > 0.05). However, several dietary differences across the diffusion outcomes were observed on PND 58 (p < 0.05) indicating dietary impacts on brain microstructure. Minimal dietary differences were observed from myelin water fraction imaging at either time-point. Overall, sialyllactose supplementation had no effects on learning and memory as assessed by novel object recognition, but may influence temporally-dependent aspects of brain development.

Immunoregulation of bovine lactoferrin together with osteopontin promotes immune system development and maturation.

The immune system of infants is partly weak and immature, and supplementation of infant formula can be of vital importance to boost the development of the immune system. Lactoferrin (LF) and osteopontin (OPN) are essential proteins in human milk with immunoregulation function. An increasing number of studies indicate that proteins have interactions with each other in milk, and our previous study found that a ratio of LF : OPN at 1 : 5 (w/w, denoted as LOP) had a synergistic effect on intestinal barrier protection. It remains unknown whether LOP can also exert a stronger effect on immunoregulation. Hence, we used an in vitro model of LPS-induced macrophage inflammation and in vivo models of LPS-induced intestinal inflammation and early life development. We showed that LOP increased the secretion of the granulocyte-macrophage colony-stimulating factor (132%), stem cell factor (167%) and interleukin-3 (176%) in bone marrow cells, as well as thymosin (155%) and interleukin-10 (161%) in the thymus, more than LF or OPN alone during development, and inhibited changes in immune cells and cytokines during the LPS challenge. In addition, analysis of the components of digested proteins in vitro revealed that differentially expressed peptides may provide immunoregulation. Lastly, LOP increased the abundance of Rikenellaceae, Muribaculum, Faecalibaculum, and Elisenbergiella in the cecum content. These results imply that LOP is a potential immunomodifier for infants and offers a new theoretical basis for infant formula innovation.

Cow's milk-based infant formula supplements in breastfed infants and primary prevention of cow's milk allergy: A commentary of the Committee on Nutrition of the French Society of Pediatrics

The role of nutritional interventions for the primary prevention of cow's milk allergy (CMA) remains debated as well as the role of early introduction of allergenic foods, which is largely encouraged from the beginning of complementary feeding. Considering the introduction of cow's milk protein (CMP), current recommendations suggest avoidance of any cow's milk formula (CMF) supplements in breastfed infants in the maternity ward. By contrast, based on poor evidence, some authors support systematic supplements of CMP in breastfed children at risk of allergy from the first week of life. The Committee on Nutrition of the French Society of Pediatrics considers that such a proposal requires more clinical studies and mainly randomized and placebo-controlled clinical trials before becoming a recommendation.

Trackability of proteins from probiotic Bifidobacterium spp. in the gut using metaproteomics

Abstract Beneficial effects of Bifidobacterium spp. on gut microbiota development and infant health have been reported earlier. Therefore, supplementation of infant formulas with probiotic bifidobacteria, as well as prebiotics stimulating bifidobacterial growth, has been proposed. Here, we studied the faecal microbiome of infants supplemented with specialized nutrition, of which some received a standard amino acid-based formula (AAF) and others an AAF with a specific mixture of prebiotics and a probiotic (synbiotics) using metaproteomics and 16S rRNA gene sequencing. Faecal samples were taken at baseline, as well as after 6 and 12 months fed with the specialized formula. The aim was to compare microbial differences between infants treated with the standard AAF and those who received the additional synbiotics. Our findings show that infants who received AAF with synbiotics have significantly higher levels of Bifidobacteriaceae DNA as well as significantly increased levels of Coriobacteriaceae proteins, over time. Moreover, at visit 12 months higher levels of some bifidobacterial carbohydrate-active enzymes, known to metabolize oligosaccharides, were observed in the synbiotic group compared to the non-synbiotic group. The results indicate that the synbiotics provided in our study are trackable in faecal samples on the DNA and protein level during the intervention period.

Ethnopharmacology of Botanical Galactagogues and Comprehensive Analysis of Gaps Between Traditional and Scientific Evidence

The functional potential of plants and plant-based components have been used in different cultures since time immemorial to promote milk production in women. Conditions like agalactia or insufficient production of breast milk due to various barriers intended to be a greater risk of weight loss for neonates and prompting for supplementation of infant formula. Multiple plants are traditionally used worldwide as galactagogues during the lactation period. This study aims to extract information on traditionally used galactagogue plants and compare their ethnopharmacological evidence with scientific evidence. This will help to understand the gaps in the ethnopharmacological and scientific data and thus provide future research information.Information on traditional and scientific studies was collected and analyzed on galactagogues. The ethnopharmacological data of the focused plant species were analyzed for part used, formulations, and region of its uses. All cultures traditionally used natural products as galactagogue from times immemorial, and due to scientific advances, these have also been seen as commercial products. However, because of the limited studies, it is of interest to standardize the doses, and composition of bioactive components and study the mechanism of action, its side effects, and interaction with food. This is a forward-looking research area that could be projected for manufacturing herbal formulations for lactating mothers.

Effect of Probiotic-Fortified Infant Formula on Infant Gut Health and Microbiota Modulation.

Compared to infant formula, breast milk is the best source of nutrition for infants; it not only improves the neonatal intestinal function, but also regulates the immune system and gut microbiota composition. However, probiotic-fortified infant formula may further enhance the infant gut environment by overcoming the limitations of traditional infant formula. We investigated the probiotic formula administration for one month by comparing 118 Korean infants into the following three groups: infants in each group fed with breast milk (50), probiotic formula (35), or placebo formula-fed group (33). Probiotic formula improved stool consistency and defecation frequency compared to placebo formula-fed group. The probiotic formula helped maintaining the level of secretory immunoglobulin A (sIgA), which had remarkably decreased over time in placebo formula-fed infants (compared to weeks 0 and 4). Moreover, probiotic formula decreased the acidity of stool and considerably increased the butyrate concentration. Furthermore, the fecal microbiota of each group was evaluated at weeks 0 and 4. The microbial composition was distinct between each groups, and the abundance of health-promoting bacteria increased in the probiotic formula compared to the placebo formula-fed group. In summary, supplementation of probiotic infant formula can help optimize the infant gut environment, microbial composition, and metabolic activity of the microbiota, mimicking those of breast milk.

Functional effects of human milk oligosaccharides (HMOs)

ABSTRACT Human milk oligosaccharides (HMOs) are the third most important solid component in human milk and act in tandem with other bioactive components. Individual HMO levels and distribution vary greatly between mothers by multiple variables, such as secretor status, race, geographic region, environmental conditions, season, maternal diet, and weight, gestational age and mode of delivery. HMOs improve the gastrointestinal barrier and also promote a bifidobacterium-rich gut microbiome, which protects against infection, strengthens the epithelial barrier, and creates immunomodulatory metabolites. HMOs fulfil a variety of physiologic functions including potential support to the immune system, brain development, and cognitive function. Supplementing infant formula with HMOs is safe and promotes a healthy development of the infant revealing benefits for microbiota composition and infection prevention. Because of limited data comparing the effect of non-human oligosaccharides to HMOs, it is not known if HMOs offer an additional clinical benefit over non-human oligosaccharides. Better knowledge of the factors influencing HMO composition and their functions will help to understand their short- and long-term benefits.

Effect of antenatal milk expression education on lactation outcomes in birthing people with pre-pregnancy body mass index ≥25: protocol for a randomized, controlled trial

BackgroundBirthing people with pre-pregnancy body mass indices (BMIs) ≥ 25 kg/m2, particularly those without prior breastfeeding experience, are at increased risk for suboptimal lactation outcomes. Antenatal milk expression (AME) may be one way to counteract the negative effects of early infant formula supplementation common in this population.MethodsThis ongoing, randomized controlled trial in the United States evaluates the efficacy of a telelactation-delivered AME education intervention versus an attention control condition on lactation outcomes to 1 year postpartum among 280 nulliparous-to-primiparous, non-diabetic birthing people with pre-pregnancy BMI ≥ 25 kg/m2. The assigned study treatment is delivered via four weekly online video consultations between gestational weeks 37–40. Participants assigned to AME meet with study personnel and a lactation consultant to learn and practice AME. Instructions are provided for home practice of AME between study visits. Control group participants view videos on infant care/development at study visits. Participants complete emailed surveys at enrollment (340/7–366/7 gestational weeks) and 2 weeks, 6 weeks, 12 weeks, 6 months, and 12 months postpartum. Surveys assess lactation and infant feeding practices; breastfeeding self-efficacy, attitudes, and satisfaction; perception of insufficient milk; onset of lactogenesis-II; lactation support and problems; and reasons for breastfeeding cessation. Surveys also assess factors associated with lactation outcomes, including demographic characteristics, health problems, birth trauma, racial discrimination, and weight stigma. Health information and infant feeding data are abstracted from the pregnancy and birth center electronic health record. Milk samples are collected from the intervention group at each study visit and from both groups at each postpartum follow-up for future analyses. Qualitative interviews are conducted at 6 weeks postpartum to understand AME experiences. Primary outcomes of interest are breastfeeding exclusivity and breastfeeding self-efficacy scores at 2 weeks postpartum. Outcomes will be examined longitudinally with generalized linear mixed-effects modeling.DiscussionThis is the first adequately powered trial evaluating the effectiveness of AME among U.S. birthing people and within a non-diabetic population with pre-pregnancy BMI ≥ 25 kg/m2. This study will also provide the first evidence of acceptability and effectiveness of telelactation-delivered AME.Trial registrationClinicalTrials.gov: NCT04258709.

The Effect of Human and Bovine Milk Osteopontin on Intestinal Caco-2 Cells: A Transcriptome Comparison

Osteopontin (OPN) is a multifunctional protein abundantly present in human milk, whereas the concentration is significantly lower in bovine milk. Human and bovine milk OPN are structurally similar and both proteins resist gastric digestion and reach the intestines in a bioactive form. Intervention studies have indicated the beneficial effects of supplementing infant formula with bovine milk OPN and several in vivo and in vitro studies have shown that bovine milk OPN positively influences intestinal development. To investigate the functional relationship, we compared the effect of simulated gastrointestinal digested human and bovine milk OPN on gene expression in Caco-2 cells. After incubation, total RNA was extracted and sequenced and transcripts were mapped to the human genome. Human and bovine milk OPN regulated the expression of 239 and 322 genes, respectively. A total of 131 genes were similarly regulated by the OPNs. As a control, a whey protein fraction with a high content of alpha-lactalbumin had a very limited transcriptional impact on the cells. Enrichment data analysis showed that biological processes related to the ubiquitin system, DNA binding, and genes associated with transcription and transcription control pathways were affected by the OPNs. Collectively, this study shows that human and bovine milk OPN have a significant and highly comparable effect on the intestinal transcriptome.

Characterization and bioavailability of a novel coenzyme Q10 nanoemulsion used as an infant formula supplement

Supplementation with Coenzyme Q10 (CoQ10), in patients with its deficiency, has greater odds of success if the treatment is carried out early with an appropriate formulation. For neonatal CoQ10 deficiency, infant formula supplementation could be an attractive option. However, solid CoQ10 cannot be solubilized or dispersed in milk matrix leading to an inefficient CoQ10 dosage and poor intestinal absorption. We developed and characterized a high-dose CoQ10 oil-in-water (O/W) nanoemulsion suitable to supplement infant formula without modifying its organoleptic characteristics. CoQ10 powder and soy lecithin were solubilized in an oil phase consisted of Labrasol® and LabrafacTM. The aqueous phase was Tween 80, TPGS, methylparaben and propylparaben. O/W nanoemulsion was prepared by adding dropwise the oil phase to the aqueous phase under stirring to a final concentration of CoQ10 9.5 % w/w followed by ultrasonic homogenization. Pharmacotechnical parameters were determined. This formulation resulted to be easily to be dispersed in milk matrix, stable for at least 90 days, with no cytotoxicity in in vitro assays, and higher bioavailability than CoQ10 powder. CoQ10 nanoemulsion supplementation in the infant formula facilitates the individualized administration for the child with accurate dosage, overcome swallowing difficulties and in turn could increase the treatment adherence and efficacy.

Determination of vitamins B1 and B6 in infant formula and food supplement samples using magnetic layered double hydroxide nanoadsorbent before liquid chromatography-tandem mass spectrometry

A magnetic Mg/Fe layered double hydroxide (magnetic LDH) nanocomposite was successfully prepared and used as a suitable adsorbent to simultaneously extract vitamins B1 and B6 from complex infant formula and food supplements. The important parameters affecting the extraction were optimized and the final parameters are as follows; the amount of adsorbent (20 mg), extraction time (6 min), elution time (8 min), and elution solvent ethanol in 750 µL. As-prepared nanoadsorbent was successfully in magnetic solid phase extraction set-up with no hazardous solvents and extraction performed in 6 min. The structure of nanoadsorbnet is lamellar, providing a wide surface to extract the analytes. The quantification of the analytes was accomplished using the liquid chromatography-tandem mass (LC/MS-MS) technique. Under optimum extraction conditions, the linearity ranged from 4 to 1000 ng/mL (vitamin B1) and 20 to 1000 ng/mL (vitamin B6), and the correlation of coefficients (R2) was obtained better than 0.99. The intra-day (n = 3) and inter-day precisions (n = 3 working days) calculated in the form of percent relative standard deviations (%RSDs) were obtained below 10.20%. The proposed method was successfully practiced for analyzing vitamins B1 and B6 in several brands of infant formulas and food supplements.

Health Effects of Infant Formula Supplemented with Probiotics or Synbiotics in Infants and Toddlers: Systematic Review with Network Meta-Analysis

Supplementation of infant and follow-up formula with probiotics or synbiotics has become a common practice. In 2011 and 2017, the evidence regarding the impact of these interventions was analysed systematically. Recently new evidence was published. To evaluate through a systematic review with network meta-analysis the evidence on the impact of infant formula supplemented with probiotics or synbiotics for healthy infants and 36-month-old toddlers. RCTs published between 1999-2019 for infant formulas supplemented with probiotics alone or synbiotics in healthy infants and toddlers were identified. Data analysis included clinical (gastrointestinal symptoms, risk reduction of infectious diseases, use of antibiotics, weight/height gain and frequency of adverse events) and non-clinical outcomes (changes in faecal microbiota and immune parameters). A random effect model was used. Hedges' standard mean difference (SMD) and risk ratio (RR) were calculated. Rank analysis was performed to evaluate the superiority of each intervention. Twenty-six randomised controlled trials with 35 direct comparisons involving 1957 children receiving probiotic-supplemented formula and 1898 receiving control formula were reviewed. The mean duration of intervention was 5.6 ± 2.84 months. Certain strains demonstrated a reduction in episodes of colic, number of days with fever and use of antibiotics; however, there was considerable heterogeneity which reduced the level of certainty of effect. No significant effects were observed on weight, height or changes in faecal proportions of Bifidobacteria, Lactobacillus, Bacteroides or Clostridia. Although there is some evidence that may support a potential benefit of probiotic or synbiotic supplementation of infant formulas, variation in the quality of existing trials and the heterogeneity of the data preclude the establishment of robust recommendations.

The Immunological Role of Milk Fat Globule Membrane.

Human milk is the ideal food for newborns until the age of six months. Human milk can be defined as a dynamic living tissue, containing immunological molecules, such as immunoglobulins, supra-molecular structures, such as the milk fat globule membrane (MFGM), and even entire cells, such as the milk microbiota. The milk composition changes throughout lactation to fulfill the infant's requirements and reflect the healthy/disease status of the lactating mother. Many bioactive milk components are either soluble or bound to the MFGM. In this work, we focus on the peculiar role of the MFGM components, from their structural organization in fat globules to their route into the gastrointestinal tract. Immunometabolic differences between human and bovine MFGM components are reported and the advantages of supplementing infant formula with the MFGM are highlighted.

Supplementation of Infant Formula with Bioactive Lipids Enhances Lung Development and Prevents Necrotizing Enterocolitis-Induced Lung Injury

Supplementation of Infant Formula with Bioactive Lipids Enhances Lung Development and Prevents Necrotizing Enterocolitis-Induced Lung Injury

Exploring the Potential of Human Milk and Formula Milk on Infants’ Gut and Health

Early-life gut microbiota plays a role in determining the health and risk of developing diseases in later life. Various perinatal factors have been shown to contribute to the development and establishment of infant gut microbiota. One of the important factors influencing the infant gut microbial colonization and composition is the mode of infant feeding. While infant formula milk has been designed to resemble human milk as much as possible, the gut microbiome of infants who receive formula milk differs from that of infants who are fed human milk. A diverse microbial population in human milk and the microbes seed the infant gut microbiome. Human milk contains nutritional components that promote infant growth and bioactive components, such as human milk oligosaccharides, lactoferrin, and immunoglobulins, which contribute to immunological development. In an attempt to encourage the formation of a healthy gut microbiome comparable to that of a breastfed infant, manufacturers often supplement infant formula with prebiotics or probiotics, which are known to have a bifidogenic effect and can modulate the immune system. This review aims to elucidate the roles of human milk and formula milk on infants’ gut and health.

Structured antenatal milk expression education for nulliparous pregnant people: results of a pilot, randomized controlled trial in the United States

BackgroundHand-expression, collection, and storage of breast milk during pregnancy (i.e., antenatal milk expression or AME) is a safe, potentially effective practice to reduce early, undesired infant formula supplementation among women with diabetes. The feasibility and potential impact of AME on lactation outcomes in the United States (U.S.) and among non-diabetic birthing people is unknown.MethodsThe purpose of this study was to examine the feasibility of a structured AME intervention among nulliparous birthing people in the United States. We recruited 45 low-risk, nulliparous individuals at 34–366/7 weeks of gestation from a hospital-based midwife practice. Participants were randomized to AME or a control group receiving lactation education handouts. Interventions were delivered at weekly visits at 37–40 weeks of pregnancy. The AME intervention involved technique demonstration and feedback from a lactation consultant and daily independent practice. Lactation outcomes were assessed during the postpartum hospitalization, 1–2 weeks postpartum, and 3–4 months postpartum.ResultsBetween December 2016 and February 2018, 63 individuals were approached and screened for eligibility, and 45 enrolled into the study (71%). Of 22 participants assigned to AME, 18 completed at least one AME study visit. Participants reported practicing AME on at least 60% of days prior to their infant’s birth. Most were able to express milk antenatally (15/18), more than half collected and froze antenatal milk (11/18), and 39% (7/18) supplemented their infants with antenatal milk after birth. No major problems were reported with AME. Perinatal and lactation outcomes, including infant gestational age at birth, neonatal intensive care unit admissions, delayed onset of lactogenesis II, and use of infant formula were similar between AME and control groups. Among participants in both groups who were feeding any breast milk at each assessment, breastfeeding self-efficacy increased and perceptions of insufficient milk decreased over the postpartum course.ConclusionsIn a small group of nulliparous birthing people in the U.S., AME education and independent practice beginning at 37 weeks of pregnancy was feasible. In some cases, AME provided a back-up supply of milk when supplementation was indicated or desired. The relationship between AME and lactation outcomes requires further study with adequately powered samples.Trial RegistrationThis trial was retrospectively registered at ClinicalTrials.gov on May 11, 2021 under the following registration ID: NCT04929301. https://clinicaltrials.gov/ct2/show/NCT04929301.

Using Glucose Gel to Reduce Neonatal Hypoglycemia and Promote Breastfeeding

Neonatal hypoglycemia may occur as neonates transition to the extrauterine environment. Infant formula supplementation and intravenous dextrose are the most frequent treatments for newborns with hypoglycemia. These interventions interrupt exclusive breastfeeding and disrupt maternal-neonatal bonding, as a higher level of care is often required. AWHONN identifies breastfeeding as one of the most important health behaviors affecting the morbidity and mortality among women and children. Nurse residents noted evidence that revealed buccal glucose gel has been used to reverse hypoglycemia in combination with breastfeeding in neonates and did not require NICU admission. This evidence prompted an evidence-based practice project in a 47-bed obstetric unit across two network campuses, which asked, “In neonates at risk for hypoglycemia, does the use of glucose gel compared with no glucose gel reduce the percentage of neonates admitted to the NICU for the treatment of hypoglycemia?”

Brain-immune-gut benefits with early life supplementation of milk fat globule membrane.

The milk fat globule membrane (MFGM) has been recognized as a milk component for more than 60 years, but its exact benefits remain unknown. Research on human MFGM has revealed that the membrane holds a host of bioactive components with potential benefits for the brain–immune–gut (BiG) axis in early life. Gangliosides and sphingomyelin, components within the MFGM, have been included in infant formulas for many years. Recent advancements in dairy milk processing have allowed the successful separation of MFGM from bovine milk, enabling it to be used for supplementing infant formulas. Evidence indicates the potential benefits of MFGM in early life supplementation, including better cognitive development, reduction of infection risks, and modulation of the gut microbiome. However, larger and more robust randomized trials are needed, in addition to long‐term outcome data beyond the infancy period.