The Milk Supply Equation

Home , Breast development, Lactation, Puberty

The Making of a Milk Factory

Objectives . List the major factors necessary for good milk production . Describe the role of the placenta in mammary gland development during pregnancy. . Explain two ways that environmental The Making of a Milk Factory contaminants may interfere with reproductive hormones © 2019 Lisa Marasco MA, IBCLC, FILCA

Hormones and Receptors The Milk Supply Equation

SUFFICIENT GLANDULAR TISSUE + INTACT NERVE PATHWAYS & DUCTS + ADEQUATE HORMONES & RECEPTORS + ADEQUATE LACTATION-CRITICAL NUTRIENTS + ADEQUATE & EFFECTIVE MILK REMOVAL (Good management, effective baby) = GOOD MILK SUPPLY Directors of the process

Important players by stage Hormones important to milk production

Puberty Pregnancy Lactation Insulin Estrogen Estrogen Cortisol Lactogenic Progesterone Progesterone Complex HPL Prolactin Chorionic Gonadotropin Maintains metabolism; Growth Hormone Growth Hormone Thyroxine - Supports & directs IGF-1 IGF-1 PTPRF pituitary Prolactin Prolactin Insulin Insulin Regulate mammary blood Cortisol Cortisol PTHrP - flow; calcium transport and Thyroxine Thyroxine homeostasis PTHrP PTHrP Oxytocin Oxytocin - milk delivery With permission: Andaluz Waterbirth Center

Hormone receptors 101 Hormone Receptors 201

Hormones- travel thru blood or synthesized locally Dynamic:  Expressed by genes ER α, β Expressed PR α, β R  Up & down regulation where and PRLR L, S Can be resistant as needed OTR H Can be hypersensitive Can be influenced positively or negatively by other hormones Receptors: Integral to hormone function, located on “target tissues,” on or in cells Can be influenced by environment

The Big Construction Picture

PR-A Phase I ER PR-B ERα Series of stages of organo-genesis lasting until ERβ PRLrL adulthood that are irreversible (fetal through puberty) PR-B Phase II Series of changes involving growth and secretory differentiation of the lobulo-alveolar system that are reversible Estrogen Prolactin Progesterone (Preg, lactation, involution)

Horseman, N. D. (1999). Prolactin and mammary gland development. J Mammary Gland Biol Neoplasia, 4(1), 79-88. Hormones can have multiple receptors for different effects

Fat Pad: the scaffolding of breast development Sufficient lactation tissue starts during Fetal Development

Primitive framework

Pubertal 1 Development Puberty 2 3 Development of fad pad 4 Elongation of ducts into pad Growth velocity Skeleton team of alveoli vs 5 Tanner stages Tanner Staging

Marshall, W., & Tanner, J. (1969). Variations in pattern of pubertal changes in girls. Archives of Disease in Childhood, 44(235), 291. Pubertal breast growth window ~10 yrs to 14-16 yrs

The completed breast End of Puberty: Completion of the Milk Factory clavicle Vertical span 1 ~ ribs 2 to 6 Basic structure is in place 2 Awaiting start-up 3 Horizontal span 4 ~midaxillary 5 to sternum 6

~50% glandular up to age 35

Shermak, 2010

Phase II Starts with a new pregnancy Pregnancy hormones Pituitary: “Mammotropic” Prolactin Proliferation hormones synthesized Milk making locally in breast Thyroid: cells multiply Prolactin, Progesterone, Thyroxine Estrogen, relaxin, epidermal GF, PTHrP

Differentiation Placental: Milk-making cells enlarge and mature, HPL, HCG, Ovarian: developing Terminal estrogen, Estrogen End Buds (TEBs) progesterone Progesterone

PTHrP - an unsung hero Pregnancy Development: The Critical Role of the Placenta *Present in mammary gland, human milk, mother and baby’s blood Progesterone after 3mo all from placenta Glandular development: Human Placental Lactogen Stimulates mammary branching morphogenesis (HPL) only produced in Essential for proper development placenta Lactation: Level of HPL correlates most Increases Ca+ transport from blood to milk closely with breast volume change Regulates mammary blood flow Regulates myoepithelial cell tone

illustration courtesy of T. John Martin FRS

Influence of multiples pregnancy Normal Pregnancy Development

Image courtesy of Triumph International and bubhub.com.au General signs of successful mammogenesis: More placenta = more mammary gland growth Increased breast sensitivity, nipple changes, enlargement, expressible colostrum at end of pregnancy

Ukrainian research 245 VIRGINS AND NULLIPAROUS WOMEN- OBSERVATIONS:

Development occurs at an uneven rate

Some parts of breast mature while others lag in intermediate stage

During pregnancy these parts continue uneven growth, and not all are “sufficiently complete” by labor

Conclusion: This may contribute to some cases of “primary hypogalactia” in first time mothers

Used with permission Morozova, M. G. (1961). [The significance of underdeveloped tissue of the mammary gland in the development Hennighausen, L., & Robinson, G. W. (2005). Information networks of the so-called primary hypolactation.]. Akush Ginekol (Mosk), 37, 43-50. in the mammary gland. Nat Rev Mol Cell Biol, 6(9), 715-725.

More Ukrainian research Findings

~1:1 ~2:1 N= 58 pregnant & 21 pp c-sect non-lactating Fat progressively displaced by lactating glandular & connective tissue  Measured changes in mammary gland composition during pregnancy (started at 6- 12wks gestation)  Classified women by prevalence of glandular, Speed and intensity of changes varied mixed or adipose in ≥ 3 of 4 quadrants “Proportion of secretory and fat tissues in the breasts can be used for prognosis of lactation activity….”

Morozova N, OM P. Clinical ultrasonographic correlations of lactation. Pediatria, akusherstvo ta gynekologia. 1997(5):64-65. Morozova N, OM P. Clinical ultrasonographic correlations of lactation. Pediatria, akusherstvo ta gynekologia. 1997(5):64-65. Ramsay et al. (2005). Anatomy of the lactating human breast redefined with ultrasound imaging. Journal of Anatomy, 206(6), 525-534.

Influence of Age Anatomy of a lactating breast

Glandular tissue gradually regresses, Parenchyma replaced by fatty/stromal tissue Glandular portion Left: 63% ± 9% (range 46-83%) Right: 65 ± 11% (range 45-83%) Interesting side notes: Age ≥ 30 Large proportion of glandular associated with delayed lactation tissue located within 30mm Nommsen-Rivers, 2010 of nipple base Stroma Milk intake decreased 25g for each 5-year Connective tissue increment of age Marquis, 2002 Adipose tissue Ligaments

Ramsay et al. (2005). Anatomy of the lactating human breast redefined with ultrasound imaging. Journal of Anatomy, 206(6), 525-534.

Anatomy of a lactating breast Phase II end: Involution

Dismantling and disposal of equipment Macias 2012 no longer needed Remodeling Ramsay et al. (2005). Anatomy of the lactating human breast redefined H Macias et al. WIREs Dev Biol 2012. with ultrasound imaging. Journal of Anatomy, 206(6), 525-534. DOI: 10.1002/ wdev.35, Copyright © 2012 Wiley Periodicals, Inc.

Stages of postnatal mammary gland development

In human women, lasts up to 2 weeks

Illustration depicting the two Life cycle of the breast in reproduction stages of mammary gland involution. Upon weaning, the gland is remodeled back Winter: Dormant breast to its prepregnancy state.

Stage 1 is reversible and is regulated largely by STAT3 Fall: Spring: Involution & Pregnancy Stage 2 is irreversible and “remodeling” blossoming characterized by alveolar of the breast of breast collapse and adipocyte differentiation

Influence of parity

Glands from parous animals reacted more robustly to a subsequent pregnancy

dos Santos, et al. (2015). An epigenetic memory of pregnancy in Used with permission. the mouse mammary gland. Cell Reports, 11(7), 1102-1109. http://www.veltmaatlab.net/research.html

Response to pseudo-pregnancy hormones after 6 & 12 days tx Factors that can disrupt development: Construction gone awry

“Pregnancy leaves an epigenetic memory

Effects of uteroplacental insufficiency O'Dowd et al, 2008. Progesterone Tx rescues mammary impairment L6 Mammary wt ↓, Control, Restricted, Restricted + P Reduced Litter to Wlodek, 2009 PTHrP ↓ Na/K ↑ . Uterine restriction induced GD18 Sham Control 5 pups after birth Pup milk intake ↓ . Progesterone 1mg/kg on GD20 Normal levels, .Restricted- less alveoli, fewer blood Sham Control lactation vessels, more adipose stroma control

Maternal mammary PTHrP content was correlated with control milk lactose concentration, which directly relates to volume

GD22: BWs ↓ 16%, GD 20 Lier # ↓ 25%; litter size ↓ 36% control P ↓ 46%, α-lac mRNA control Restricted Group ↑ 113% L6 Mammary wt ↓, milk lactose ↓, PTHrP ↓ ↓, control Day 22 Ligation GD 18 birth Premature Lact II triggered Na/K ↑ Subsequent involution Pump milk intake ↓ control Used with permission of Dr. Paul Kenyon Growth restricted 28% Day 22 *Milk production gradually picked up for RG birth Body wt reduct ↑ to 36%

Impact of pregnancy problems Risk factors  Effects of early delivery on mammary growth

 Baby SGA/IUGR  Hypertension  Implications of SGA baby for placental function

Impact of pregnancy hypertension Pregnancy hypertension

PIH is associated with delayed Placentas smaller, calcifications, vascular lactogenesis (Salahudeen 2013) insufficiency (Majumdar, 2005; Goswami 2012; Naharaet 2013) Mothers with gestational hypertension Hypertensive rats had reduced PTHrP, impaired have more difficulties maintaining mammary development, ↓ milk delivery (Wlodek, exclusive breastfeeding and shorter 2003) duration after 6 mos (Strapasson 2018) Timing of onset? Women with hypertensive disease of pregnancy bfd significantly less often, esp. w/HELLP (Leeners, 2005)

PIH and prolactin Implications of antenatal corticosteroids Study followed women at risk for delivering <34wks PREGNANCY 20-23 24-27 28-31 32-35 36-39 40-42 weeks weeks weeks weeks weeks weeks Normotensive 92ng/mL 128ng/mL 149ng/mL 225ng/mL 205ng/mL 229ng/mL Observations: N=121 29-172 49-266 69-440 70-566 65-584 64-496 Pre-eclampsia 155ng/mL 183ng/mL 178ng/mL  Women >28 wks who delivered within 2 days of 27-465 47-414 63-326 treatment produced higher volumes of milk than Essential 169ng/mL 180ng/mL 171ng/mL 149ng/mL those delivered between 2-9 days post tx (primary) 50-375 85-444 52-420 104-219 hypertension  Milk volume increased significantly with advancing gestational age at delivery

Yuen, B. H., Cannon, W., Woolley, S., & Charles, E. (1978). Maternal plasma and amniotic fluid prolactin levels in normal and hypertensive pregnancy.  Rare: Trigger of premature Lactogenesis II

Effect of preterm birth and antenatal corticosteroid treatment on lactogenesis II in women (Henderson 2008)

Endocrine May… Disruptors Mimic endogenous hormones Include Antagonize endogenous hormones Alter synthesis & metabolism of natural hormones Modify hormone receptor levels Block normal binding of hormones to their receptors Bind preferentially to receptors Markey 2002

Altered Cell response

Skin exposure: Inhalation st Cosmetics Exposure: 21 century Lactation Challenges Body creams PAHs Deodorants PBDEs Shampoos Plasticisers Perfumes Heavy metals

Oral Exposure: Food Accumulation of fat-soluble contaminants chemicals: Plasticisers (DDT, DDE, PCBs, PBDEs) PAHs Organochlorines Pesticides or fungicides Heavy metals

Transfer of fat-soluble chemicals via breastmilk Transfer from mother to fetus, amniotic fluid or both

Every day routes of toxin exposure Lee, S., & Kelleher, S. L. (2016). Biological underpinnings of breastfeeding challenges: the role of genetics, diet, and environment on lactation physiology. Am J Physiol Endocrinol Metab, 311(2), E405-422.

Environmental contaminant exposure?

“Standard lactation guidelines are based on the assumption hat virtually any woman can breastfeed normally” Concerns started w/ observations of contaminated milk and lower bfg durations “It isn’t clear whether altered development of the mammary gland itself or disruptions in hormonal status are responsible for the negative impacts on lactation…”

ncbi.nlm.nih.gov/pmc/articles/PMC5226708/pdf/ehp.125-A17.pdf Knight C, Sorenson A. Windows in early mammary development: critical or not? Reproduction 2001; 122:337-345

Environmental Contaminants- a neglected threat? Effects of early life EDC Exposure [rats]

Effects of Xenobiotics on milk secretion Neville & Walsh 1995

Hypothesized 3 modes of interference Mammary development via estrogenic disruption DIRECTLY on milk secretion process  Could open paracellular pathway (tight junctions) INDIRECTLY on lactational hormonal milieu or delivery of milk substrates

Rudel, et al (2011). Environmental exposures and mammary gland development: state of the science, public health implications, and research recommendations.

Estrogen-like endocrine disrupting chemicals DDE & Lactation Studies Study Year Location Findings Chemical / Group Sub-types Effects Rogan & Gladen 1985 North Carolina “women with higher DDE levels do not breastfeed as long.” Top 5% had median duration of 9wks vs DDT DDE (dichloroethylene)- estrogen mimicker/blocker 35wks in lowest 6% (Dichlorodiphenyltrichloroethane) metabolite of DDT Abnormal breast development Gladen & Rogan 1995 Comarca Lagunera, Found decrease in duration of lactation among (Den Hond, 2002) Mexico Mexican women who had previously bfd. Compared to US population study. IMS most common reason for weaning. Dioxins TCDD, PCB, PBE, CDE, CDF, Block ERs, anti-estrogenic PCAH characteristic; retarded breast Karmaus, et al 2005 Michigan DDE & PCB estimated by milk levels. Breastfeeding development (Schoeters, 2008) Great Lakes angler duration shorter when concentrations higher: families 13wks for > 10μ/L vs 21.7 wks for lower DDE BPA Precocious puberty Binds to thyroid hormone Guillette, et al 2006 Yaqui Valley, Sonora, Mex Abnormal breast development (Bisphenol –A) Exposed since 50’s receptor, may reduce T3 Cupul-Uicab, et al 2008 Tapachula, Chiapas, Did not find association w/ shorter lactation. Used dimethyl, diethyl, dibutyl, and “defeminization;” Mexico mother-son pairs only. Excluded higher age, Phthalate esters 40 yrs exposure preeclampsia, GD, PIH, BW <2500gm. di-(2-ethylhexyl); metabolite premature breast mono-(2-ethylhexyl) development (Colon, 2000) Weldon 2006 Agricultural California- Levels of DDE presented in study are lower than in 600 low income Mexican- previous studies “which may explain the lack of a Am mothers significant association with lactation.” Lead Abnormal breast development Weldon 2010 366 Mex-Am mothers in Shorter duration found in women who previously California bfd only, attributed to lactation hx bias. Some Roy, 2009; Colon, 2000; Schoeters, 2008; Den Hond, 2002 lengthened duration found in first breastfeeders.

Dioxin & Breast/Lactation Studies Study Year Location /Type Findings Mexico 2016: BPA and Breastfeeding Fenton, et al 2002 Rat Gestational exposure to TCDD before migration of TCDD mammary bud to fat pad permanently alters Odds of not breastfeeding @ 1 mo mammary epithelial development in offspring- problem occurs in stroma Vorderstrasse, et al 2004 Mice Gestational exposure. Severe defects in mammary TCDD gland due to inappropriate activation of AhR during fetal development. Diminished milk production Fenton 2006 Animal EDC (BPA, Majority of effects found after exposure during critical dioxin) Lit analysis & periods of development (fetal budding, puberty, Odds Perceived Insufficient Milk@ 1 mo summary pregnancy)

Leijs, et al 2008 Human Amsterdam cohort. Delayed initiation of breast PCDD/F, dl-PCB, development. PBDE Collins 2009 Mice & in vitro Late gestational exposure more vulnerable to TCDD disruption of alveolar development . Altered AhR → BPA concentration in urine ↓ phophorylaon of Stat5 → ↓milk protein gene induction. Lew,et al 2009 Mice Exposure in early pregnancy negatively influences TCDD/ mammary proliferation; exposure in mid-pregnancy Kasper (2016). Association of Bisphenol A Exposure with Breastfeeding and Perceived Insufficient Milk Supply in Mexican Women. Matern Child Health J, 20(8), 1713-1719. AhR activation negatively influences mammary differentiation.

New in the news: PFOAs 2016: Summary of Effects of common chemicals on mammary gland, lactation

Gestational Impairs differ- Exposure in Disrupt Activates aryl exposure → entiation of early gest or ↓Milk protein Found in mammary hydrocarbon mammary alveolar units in lact → lactation expression function receptor hypoplasia lactation insufficiency Atrazine herbicides   Dioxins (ie PCB) herbicides PFOA in preg ↑    (severe)  Bfg duraon ↓ BPA plastics    Dibutylphthalate plastics  Nonlyphenol laundry & dish  detergent Polybrominated flame retardant  diphenyl ethers Perfluorooctanoic cleaning    acid (PFOA) products, (permanently) pesticides

Source: Lee and Kelleher 2016 Romano, et al. (2016). Maternal serum perfluoroalkyl substances during pregnancy and duration of breastfeeding. Environ Res. Photo by Dollen. CC by-ND 2.0

2017: Triclosan (antibacterials) & Methyl paraben (personal care products) 2015 Testing of 3 EDCs at human low dose exposure Manservisi 2015

Pre-puberty, puberty, perinatal, lactation Tested low doses of: Findings: windows Diethylphtalate Morphological change in gland Low doses induced measurable changes in (DEP) at end of lactation histology Methylparaben Increased sensitivity of the (MPB) mammary gland to EDCs Chronic exposure of TCS from birth-lactation Triclosan (TCS) during pregnancy and caused ↑ adipose ssue ↓ glandular & Mixture of all lactation secretory tissue Olive Oil Changes in gene expression at end of lactation

Gopalakrishnan, et al. (2017). Changes in mammary histology and transcriptome profiles by low-dose exposure to environmental phenols at critical windows of development. Environ Res, 152, 233-243.

Endocrine disruptors: Primary? Pregnancy? Defining Hypoplasia What did she How did it respond start with? to pregnancy? What do we know about her own mother and gestation? In what environments has mom lived or been Damaged tissue exposed to chemically? Prior jobs? Fish from contaminated lakes? Exposures during prior pregnancy?

Fenton, S. E. (2006). Endocrine-disrupting compounds and mammary gland development: early Poor response exposure and later life consequences. Endocrinology, 147(6 Suppl), S18-24. Scant starting tissue to pregnancy

What exactly is normal, anyways? Congenital problems

Receptor deficiency Case of missing Estrogen & Progesterone receptors

Ito, et al. (2004). Mammary hypoplasia resulting from hormone receptor deficiency. Plastic and reconstructive surgery, 113(3), 975-977.

Hypomastia and Mitral Obesity: a risk factor for hypoplasia valve prolapse Rosenberg 1983

Bever Babendure, et al. (2015). Reduced MVP Hypomastia breastfeeding rates among obese mothers: a review Group A: Hypomastia N =27 13 (48%) 27 (100%) of contributing factors, clinical considerations and future directions. Int Breastfeed J, 10, 21. Group B: Controls N= 33 2 (6%) 8 (24%)

Group C: MVP N= 28 28 (100%) 17 (61%)

Impact of diet-induced obesity Flint 2005 mouse study:* Impact of Mammary tree extended full into the fat pad BUT Disrupted morphology exhibited decrease branching frequency and high-fat diet in Dramatic decrease in # impaired alveolar development in pregnancy pregnancy of intact alveolar units *induced prior to mating ↑ 5-HT indicative of inflammatory process

Kamikawa 2009 mouse study:** “Consumption of high-fat diets in pregnancy has also been determined Adipocytes/fat pad/ mammary gland were enlarged to decrease the amount of myoepithelium in the mammary gland” Hernandez et al. (2012). High Fat Diet Alters Lactation Outcomes: with abnormal stroma, signs of inflammatory process, Possible Involvement of Inflammatory and Serotonergic Pathways. PLoS One, 7(3), e32598. abnormal and reduced ductal branching, partial deficit “Consumption of high fat diet impairs mammary parenchymal in myoepithelium, increased leptin tissue and impedes its ability to synthesize and secrete milk…”

**induced in puberty, no pregnancy De Assis, S., & Hilakivi-Clarke, L. (2006). Timing of dietary estrogenic exposures and breast cancer risk. Ann N Y Acad Sci, 1089, 14-35. doi:10.1196/annals.1386.039

What about eating disorders? Peer experience: “A decrease in breast size- or if you haven’t gone through puberty yet, a halt in breast development- is normal in anorexia nervosa. With recovery, breast development will resume.”

“If you get an ED at age 14, puberty stops, and you recover at age 26, it is probably too

Knight C, Sorenson A. Windows in early mammary development: critical or not? Reproduction 2001; 122:337-345. late.”

Knight C, Sorenson A. Windows in early mammary development: critical or not? Reproduction 2001; 122:337-345

Pregnancy breast increment changes Vanky 2012 Risky Breast Shapes

More difficult Breast size increment correlates positively to classify with duration of breastfeeding… Marked

3 asymmetry 1 2 Women with no breast size change: considered Higher blood pressure “classic,” but often presents More obese without Higher fasting insulin higher dramatic

The Bottom Line: It’s all about glandular development

Having the right amounts of free hormones and functional receptors Right Place Right Time

For Fetal Development For Pubertal Development For Pregnancy Development LaPensee, E. W., & Ben-Jonathan, N. (2010). Novel roles of prolactin and estrogens in breast cancer: resistance to chemotherapy. Endocrine-Related Cancer, 17(2), R91-R107. doi: 10.1677/erc-09-0253