Fetal Mammary Gland Research Articles

Fetal Mammary Gland Development and Offspring’s Breast Cancer Risk in Adulthood

While advancements in early detection and improved access to care have significantly enhanced breast cancer survival rates, the disease remains a significant global malignancy, constituting approximately 12.5% of all new cancer cases and claiming nearly 700,000 lives in 2020. As a result, there is widespread consensus that the most sustainable solution lies in prevention. Indeed, preventive strategies, including lifestyle modifications and research into risk-reducing interventions, offer the potential to address the root causes of noncommunicable diseases such as breast cancer. While conventional wisdom has long attributed established risk factors for breast cancer to age, lifestyle, familial history, and reproductive factors, evidence highlights the maternal environment as a pivotal stage for fetal programming of disease risk, as elucidated in the developmental origins of health and disease (DOHaD) framework. Consequently, a growing body of research has been focused on elucidating epigenomic signatures that influence fetal development while shaping health outcomes and susceptibility to diseases later in life. This review aims to identify fetal mammary developmental genes that have been implicated in breast cancer etiology and the potential interplay of maternal environment in epigenetic programming of breast cancer risk in adulthood.

PSI-13 Effects of maternal nutrient restriction and melatonin supplementation on fetal mammary gland vascularity

Abstract The objective was to determine fetal mammary gland vascularity following dietary melatonin supplementation in late pregnant nutrient restricted Brangus heifers carrying female fetuses from two different seasons (spring calving, n = 12; fall calving, n = 13). Heifers were assigned one of four nutritional treatments at day 160 of gestation; adequate fed (ADQ-CON; 100% NRC; n = 6), nutrient restricted (RES-CON; 60% NRC; n = 6), adequate fed with 20 mg of melatonin (ADQ-MEL; n = 6), and nutrient restricted with 20 mg of melatonin (RES-MEL; n = 7). Fetal mammary glands were collected at day 240 of gestation following Cesarean delivery. Mammary gland vascularity was determined via immunofluorescence staining and analyzed via ANOVA. In spring calving heifers, maternal body weight was decreased (P = 0.002) in RES (528±11 kg) vs ADQ (601±11 kg) fed. Fetal body weight, fetal mammary gland weight, and mammary gland vascularity were not different (P > 0.15) among treatments. In fall calving heifers, a nutrition by treatment interaction (P = 0.048) was observed for maternal body weight, which was increased in ADQ-MEL versus all groups. Fetal body weight and fetal mammary gland weight were not different (P > 0.45) among groups. In fall calving, fetal mammary gland capillary number and average size were not different (P > 0.40) among groups. However, a nutrition by treatment interaction was observed for capillary area (P = 0.05), which was increased in RES-MEL versus ADQ-MEL. In addition, a nutrition by treatment interaction was observed for capillary perimeter (P = 0.001), which was increased in RES-MEL versus all groups. Overall, maternal nutrient restriction and melatonin supplementation did not impact fetal mammary gland vascularity in spring calving heifers; however, melatonin supplementation during nutrient restriction in fall calving heifers may increase fetal mammary gland vascularity. In conclusion, seasonal differences and natural melatonin production may influence melatonin-induced changes in fetal development.

In utero exposure to thermal stress has long-term effects on mammary gland microstructure and function in dairy cattle

Earth’s rising temperature has substantial repercussions for food-producing animals by increasing morbidity and mortality, diminishing reproductive potential, and reducing productivity. In the dairy industry this equates to massive losses in milk yield, which occur when cows are exposed to heat stress during lactation or during the non-lactating period between lactations (i.e. dry period). Furthermore, milk yield is significantly lower in first-lactation heifers that experienced fetal heat stress. The mechanisms underlying intrauterine effects of heat stress on the offspring’s future lactation have yet to be fully elucidated. We hypothesize that heat stress experienced through the intrauterine environment will alter the mammary gland microstructure and cellular processes involved in cell turnover during the cow’s first lactation. Mammary biopsies were collected from first-lactation heifers that were exposed to heat stress or cooling conditions while developing in utero (IUHT and IUCL; respectively, n = 9–10). IUHT heifers produced less milk compared to IUCL. The mammary glands of IUHT heifers differed morphologically from IUCL, with the IUHT heifers having smaller alveoli and a greater proportion of connective tissue relative to their IUCL herdmates. However, intrauterine heat stress had little impact on the proliferation and apoptosis of mammary cells during lactation. Our results indicate that fetal exposure to heat stress impairs milk production in the first lactation, in part, by inducing aberrant mammary morphology. This may result from alterations in the developmental trajectory of the fetal mammary gland that persist through the first lactation rather than to alterations in the cellular processes controlling mammary cell turnover during lactation.

Histochemical properties of bovine and ovine mammary glands during fetal development.

In order to obtain more information on the development of bovine and ovine fetal mammary glands, a series of mammary glands from fetuses of different ages were analyzed. A total of 16 bovine fetuses with curved crown rump lengths ranging from 12 cm (80 days) to 75 cm (240 days) and 15 ovine fetuses ranging from 55 days to 131 days were examined. We used hematoxylin and eosin stain and Oil-Red-O stain to analyze the developmental and morphogenetic processes of mammary glands. In addition, we used immunohistochemical staining to determine the pattern of expression of cytokeratin 18 (CK18) during luminal epithelial differentiation, α-smooth-muscle actin (α-SMA) for myoepithelial differentiation, Ki-67 for cell proliferation, and estrogen receptor α (ERα). Our analyzes showed: (a) The primary mammary duct begin to proliferate in a lengthwise within the teat at 90 days in bovine fetuses and 63 days in ovine fetus; (b) luminal epithelial cells and myoepithelial cells appeared from 90 days in bovine fetuses and 63 days in ovine fetus; (c) proliferation of epithelial cells appeared to coincide with the development of the primary and secondary ducts; and (d) ERα was not found in the fetal mammary gland, but adipocytes showed the presence of ERα. Overall, these results indicate that the sequence of events in the prenatal development of the mammary gland of sheep is similar to that of cattle.

New insights into fetal mammary gland morphogenesis: differential effects of natural and environmental estrogens

An increased breast cancer risk during adulthood has been linked to estrogen exposure during fetal life. However, the impossibility of removing estrogens from the feto-maternal unit has hindered the testing of estrogen’s direct effect on mammary gland organogenesis. To overcome this limitation, we developed an ex vivo culture method of the mammary gland where the direct action of estrogens can be tested during embryonic days (E)14 to 19. Mouse mammary buds dissected at E14 and cultured for 5 days showed that estrogens directly altered fetal mammary gland development. Exposure to 0.1 pM, 10 pM, and 1 nM 17 β-estradiol (E2) resulted in monotonic inhibition of mammary buds ductal growth. In contrast, Bisphenol-A (BPA) elicited a non-monotonic response. At environmentally relevant doses (1 nM), BPA significantly increased ductal growth, as previously observed in vivo, while 1 μM BPA significantly inhibited ductal growth. Ductal branching followed the same pattern. This effect of BPA was blocked by Fulvestrant, a full estrogen antagonist, while the effect of estradiol was not. This method may be used to study the hormonal regulation of mammary gland development, and to test newly synthesized chemicals that are released into the environment without proper assessment of their hormonal action on critical targets like the mammary gland.

Nutritional plane of twin-bearing ewes alters fetal mammary gland biochemical composition and mTOR/MAPK pathway signaling.

Identifying the biochemical changes and molecular pathways that regulate fetal mammary development in response to maternal nutrition is important for understanding the link between fetal programming of mammary development and future lactation performance. Although there are published studies regarding biochemical changes in the developing mammary gland, there are currently no data on molecular pathway involvement in regulating ruminant fetal mammary development. This study investigated changes in fetal mammary biochemical indices and mechanistic target of rapamycin (mTOR)/mitogen activated protein kinase (MAPK) signaling at d 100 and 140 of gestation in an ovine model of restricted maternal nutrition. Ewes were randomly allocated to ad libitum (A) or maintenance (M) nutritional regimens, under New Zealand pastoral grazing conditions, from d 21 to 140 of pregnancy. At d 100 and 140 of pregnancy, a subgroup of twin-bearing dams was euthanized, and whole fetal mammary glands (fiber, skin, fat, and ducts) were collected. Mammary glands of fetuses carried by M-fed dams were heavier at d 100 than those of fetuses carried by A-fed dams ( = 0.03), with no difference in the abundance of mTOR/MAPK signaling proteins observed. At d 140, mammary glands of fetuses carried by M-fed dams were lighter ( = 0.07) than fetuses carried by A-fed dams because of decreased hyperplasia ( = 0.04) and hypertrophy ( = 0.09) but had increased protein synthetic capacity ( = 0.02). Increased protein synthetic capacity was associated with increased abundance of MAPK pathway signaling proteins eukaryotic intiation factor 4E (eIF4E)/eIF4E and mTOR pathway signaling proteins eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)/4E-BP1 and ribosomal protein S6 (RPS6)/RPS6 ( ≤ 0.05). Increased abundance of MAPK/mTOR pathway proteins is proposed to mediate increased protein synthetic capacity via ribosome biogenesis and the availability of factors required to initiate protein translation. The primary regulator of 4E-BP1 phosphorylation at Ser65 and RPS6 at Ser235/236 is the activated form of mTOR: mTOR. To study potential tissue-specific mTOR, mTOR abundance mammary glands, separated into parenchyma and fat pad, were collected from d 140 fetuses carried by dams fed a lucerne-based pellet diet formulated to meet 100% of the NRC-recommended maintenance requirements. Results showed that the abundance of mTOR was primarily localized to the fat pad, indicating that the fat pad plays a potential role in regulating development of the fetal mammary gland.

Estrogens in the wrong place at the wrong time: Fetal BPA exposure and mammary cancer.

Iatrogenic gestational exposure to diethylstilbestrol (DES) induced alterations of the genital tract and predisposed individuals to develop clear cell carcinoma of the vagina as well as breast cancer later in life. Gestational exposure of rodents to a related compound, the xenoestrogen bisphenol-A (BPA) increases the propensity to develop mammary cancer during adulthood, long after cessation of exposure. Exposure to BPA during gestation induces morphological alterations in both the stroma and the epithelium of the fetal mammary gland at 18 days of age. We postulate that the primary target of BPA is the fetal stroma, the only mammary tissue expressing estrogen receptors during fetal life. BPA would then alter the reciprocal stroma-epithelial interactions that mediate mammogenesis. In addition to this direct effect on the mammary gland, BPA is postulated to affect the hypothalamus and thus in turn affect the regulation of mammotropic hormones at puberty and beyond.

Abstract A136: Deconstructing stemness from the fetal mammary perspective

Abstract Somatic stem cells govern the development, maintenance and regeneration of tissues, and their dysregulation is associated with diverse pathologies including cancer. Given the significance of these cells both biologically and therapeutically, it is critical to elucidate the signaling mechanisms that dictate their behavior. Our recent work on the fetal mammary gland has dissected complementary roles for the stem cell compartment and stromal cues in fetal mammary stem cell function. We also identified significant similarities between the gene expression profiles of some of the most aggressive and untreatable forms of human breast cancer and those of the mouse fetal stem cell enriched mammary epithelial and stromal fractions. These similarities suggest that stem cell regulatory mechanisms operative during organogenesis, including those specified by the stromal niche, may serve as novel therapeutic targets in these cancers. We also discovered significant gene expression heterogeneity within the fetal stem cell compartment when analyzed at single cell resolution. While such heterogeneity likely reflects the natural diversity of stem and non-stem cell states comprising the system, it confounds the interpretation of expression profiles from mixed populations and the delineation of stem cell specific and niche specific molecular components. Our current objective is to utilize genome wide single cell RNA-Sequencing to hasten the identification of functionally relevant, stem cell specific regulators and markers in the context of this multi-cellular/multi-signal system. Among other candidates, we have identified the Cripto/GRP78 signaling axis as a critical regulator of mammary stem cell activity, and have developed novel molecular genetic tools to temporally control the activity of candidate stem cell regulators in vivo in normal and neoplastic contexts. Integration of these tools promises to deliver a new molecular understanding of stem cell biology in the mammary gland and other systems and of stem cell-like cancer cells. Citation Format: Benjamin T. Spike, Danielle D. Engle, Jennifer C. Lin, Jonathan A. Kelber, Justin La, Samantha K. Cheung, Evan Booker, Rose Rodewald, Christopher Dravis, Peter C. Gray, Geoffrey M. Wahl. Deconstructing stemness from the fetal mammary perspective. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A136.

Low-Dose BPA Exposure Alters the Mesenchymal and Epithelial Transcriptomes of the Mouse Fetal Mammary Gland

Exposure of rodent fetuses to low doses of the endocrine disruptor bisphenol A (BPA) causes subtle morphological changes in the prenatal mammary gland and results in pre-cancerous and cancerous lesions during adulthood. To examine whether the BPA-induced morphological alterations of the fetal mouse mammary glands are a) associated with changes in mRNA expression reflecting estrogenic actions and/or b) dependent on the estrogen receptor α (ERα), we compared the transcriptomal effects of BPA and the steroidal estrogen ethinylestradiol (EE2) on fetal mammary tissues of wild type and ERα knock-out mice. Mammary glands from fetuses of dams exposed to vehicle, 250 ng BPA/kg BW/d or 10 ng EE2/kg BW/d from embryonic day (E) 8 were harvested at E19. Transcriptomal analyses on the ductal epithelium and periductal stroma revealed altered expression of genes involved in the focal adhesion and adipogenesis pathways in the BPA-exposed stroma while genes regulating the apoptosis pathway changed their expression in the BPA-exposed epithelium. These changes in gene expression correlated with previously reported histological changes in matrix organization, adipogenesis, and lumen formation resulting in enhanced maturation of the fat-pad and delayed lumen formation in the epithelium of BPA-exposed fetal mammary glands. Overall similarities in the transcriptomal effects of BPA and EE2 were more pronounced in the epithelium, than in the stroma. In addition, the effects of BPA and EE2 on the expression of various genes involved in mammary stromal-epithelial interactions were suppressed in the absence of ERα. These observations support a model whereby BPA and EE2 act directly on the stroma, which expresses ERα, ERβ and GPR30 in fetal mammary glands, and that the stroma, in turn, affects gene expression in the epithelium, where ERα and ERβ are below the level of detection at this stage of development.

LACTATION BIOLOGY SYMPOSIUM: Maternal nutrition during early and mid-to-late pregnancy: Comparative effects on milk production of twin-born ewe progeny during their first lactation1,2

Studies using sheep models indicate that the fetal mammary gland is sensitive to maternal nutrition during gestation; however, results have been inconsistent and do not identify critical feeding periods. This study aimed to clarify previous findings by partitioning the period of maternal nutritional manipulation into 2 stages: early and mid-to-late pregnancy. Sixty-six twin-born, twin-bearing ewes, born to dams that were fed either submaintenance, maintenance, or ad libitum during early pregnancy (d 21 to 50 of pregnancy; SmP21-50, MP21-50, or AdP21-50, respectively) and then either maintenance or ad libitum during mid-to-late pregnancy (d 50 to 140 of pregnancy; MP50-140 or AdP50-140, respectively) were milked once a week, starting from d 7 ± 1 postpartum, for 7 subsequent weeks to enable estimation of daily milk yield and composition. Their lambs were weighed weekly. Ewes born to dams fed MP21-50 tended to have greater accumulated milk (P = 0.10), fat (P = 0.07), and NE (P = 0.06) yields over 50 d compared with ewes born to dams fed SmP21-50 and AdP21-50. In contrast, ewes born to dams fed AdP50-140 tended to have greater accumulated milk (P = 0.10) and lactose (P = 0.09) yields compared with ewes born to dams fed MP50-140. Grandoffspring birth weights were unaffected by granddam nutrition during pregnancy. Ewes born to dams fed AdP21-50 weaned lighter lambs (P = 0.05) than ewes born to dams fed AmP21-50 and tended to wean lighter lambs (P = 0.07) than ewes born to dams fed MP21-50 whereas there were no differences between the weaning weights of lambs (P = 0.43) from ewes born to dams fed AdP50-140 and MP50-140. Maintenance nutrition of dams during early pregnancy appears to be associated with an improved lactation performance of ewe offspring. Higher levels of nutrition during mid-to-late pregnancy also appears to improve the first-lactation performance of ewe offspring. Interestingly, although grandoffspring birth weights were unaffected, weaning weight appears to be influenced by granddam early pregnancy nutrition in a manner discordant with the lactational performance of their dam. Results from this study indicate that dam nutrition during early pregnancy can influence the lactational performance of ewe offspring and the BW at weaning of their grandoffspring, which may ultimately affect farmer profits. This highlights the importance of nutritional management of breeding ewes during this period.

Chitosan Biomaterials Induce Branching Morphogenesis in a Model of Tissue-Engineered Glandular Organs in Serum-Free Conditions

Glandular organs feature ramified structures that are important for regulating physiological transport. The aim of this study was to develop a biomaterial-assisted, serum-free culture system to generate branching structures in explants of glandular organs. The fetal mammary gland (MG) was selected as the model organ to study the formation of glandular structure. Among the many biomaterials tested, chitosan demonstrated a superior effect in promoting branch formation in MGs. The morphogenetic effect toward MG branching was chitosan specific and not observed with other analogs with similar chemical compositions or structures. The molecular weight and specific linkages in the chitosan polymer were important parameters in mediating the morphogenetic effect. MG explants from different anatomical locations effectively promoted structure formation. Blocking endogenous fibroblast growth factor 10 (FGF10) inhibited the morphogenetic effect of chitosan, indicating that the chitosan effect was FGF10 dependent. This work demonstrates the feasibility of creating a serum-free system that is competent in facilitating tissue morphogenesis in MG. MG tissue structure can be efficiently generated in a biocompatible system, which was assisted by biomaterials.

Bisphenol A alters the development of the rhesus monkey mammary gland

The xenoestrogen bisphenol A (BPA) used in the manufacturing of various plastics and resins for food packaging and consumer products has been shown to produce numerous endocrine and developmental effects in rodents. Exposure to low doses of BPA during fetal mammary gland development resulted in significant alterations in the gland's morphology that varied from subtle ones observed during the exposure period to precancerous and cancerous lesions manifested in adulthood. This study assessed the effects of BPA on fetal mammary gland development in nonhuman primates. Pregnant rhesus monkeys were fed 400 μg of BPA per kg of body weight daily from gestational day 100 to term, which resulted in 0.68 ± 0.312 ng of unconjugated BPA per mL of maternal serum, a level comparable to that found in humans. At birth, the mammary glands of female offspring were removed for morphological analysis. Morphological parameters similar to those shown to be affected in rodents exposed prenatally to BPA were measured in whole-mounted glands; estrogen receptor (ER) α and β expression were assessed in paraffin sections. Student's t tests for equality of means were used to assess differences between exposed and unexposed groups. The density of mammary buds was significantly increased in BPA-exposed monkeys, and the overall development of their mammary gland was more advanced compared with unexposed monkeys. No significant differences were observed in ER expression. Altogether, gestational exposure to the estrogen-mimic BPA altered the developing mammary glands of female nonhuman primates in a comparable manner to that observed in rodents.

Dam and granddam feeding during pregnancy in sheep affects milk supply in offspring and reproductive performance in grand-offspring1

In temperate climates, the cost of providing feed is greater in winter than in other seasons, causing ewes to be fed restricted rations during some periods of pregnancy. Epidemiological information indicates that undernutrition of the fetus may affect its health and performance in later life (i.e., fetal programming), and these effects may be passed between generations. The primary focus of the results presented in this paper is to examine the effects of feeding levels during pregnancy on a variety of traits from offspring at the fetal stage to 3.5 yr of age and also traits in the grand-offspring. Two studies are reported in which ewes were fed restricted diets during pregnancy, with a variety of fetal traits, offspring traits up to 3.5 yr of age, or grand-offspring traits up to 8 mo of age being measured. Study 2 also considered differences in dam size (heavy vs. light). In study 1, several fetal mammary gland measures indicated that milking ability may be enhanced in offspring from dams fed ad libitum during pregnancy. However, study 2 showed that mammary mass was greater in fetuses from dams fed at maintenance during pregnancy and that contemporaries of these fetuses produced greater protein and lactose yields in their first lactation. In the second lactation, the advantages in protein and lactose yields did not reoccur and ewes from ad libitum-fed dams produced greater fat yield. In study 2, grand-offspring whose granddams were fed at maintenance levels during pregnancy were lighter at birth in both the first and second parturitions than those whose granddams were fed ad libitum during pregnancy. First-parity grand-offspring whose granddams were fed maintenance levels during pregnancy achieved heavier BW by 40 to 50 d of age in the first lactation, which reflected the greater protein and lactose yields; however, no BW differences were present in second-parity lambs at the same age. A smaller proportion of first-parity ewe grand-offspring from heavy granddams that were fed ad libitum during pregnancy reached puberty at approximately 8 mo of age relative to the other granddam size and feeding groups. These results indicate that dam nutrition can affect the yield and composition of milk in their offspring and the BW and reproductive capability of their grand-offspring. Molecular and physiological mechanisms for these changes are being sought.

Effects of ewe size and nutrition on fetal mammary gland development and lactational performance of offspring at their first lactation1

Many environmental factors applied postnatally are known to affect milk production of the dam, but to date, the effects of different fetal environments on subsequent first lactational performance of the offspring have not been reported. Four hundred fifty heavy (H; 60.8 kg +/- 0.18) and 450 light (L; 42.5 kg +/- 0.17) dams were randomly allocated to ad libitum (A) or maintenance (M) nutritional regimens from d 21 until d 140 of pregnancy, under pastoral grazing conditions (HA, n = 151; HM, n = 153; LA, n = 155; LM, n = 153). At d 100 of pregnancy, a sub-group of twin-bearing dams was killed and fetal mammary glands collected. From 1 wk before lambing, all remaining dams were fed ad libitum until weaning. After weaning, female progeny were managed and fed under pastoral conditions as 1 group. At 2 yr of age, 72 twin-rearing ewe offspring were milked once a week for 7 wk. Fetuses from M-dams had heavier mammary glands (P = 0.03) compared with A-fetuses. Fetuses from H-dams had greater (P = 0.0008) mammary duct area compared with L-fetuses. At 2 yr of age, M-offspring had greater milk yields at d 7 (P = 0.02) and d 28 (P = 0.09) of lactation and tended to have greater accumulated milk yields (P = 0.11) compared with A-offspring. Ewes born to M-dams showed greater lactose percentage at d 14 (P = 0.002), d 21 (P = 0.06), and d 28 (P = 0.07) of lactation and greater (P = 0.049) accumulated lactose yields and CP (P = 0.06) yields compared with A-offspring. Ewes born to H-dams displayed greater milk yields at d 14 (P = 0.08) and d 21 (P = 0.02) and had greater accumulated milk yield (P = 0.08) and lactose yield (P = 0.04) compared with L-offspring. Lambs born to M-offspring were heavier at birth (P = 0.02) and grew faster until weaning (P = 0.02), matching the milk yield and composition data, compared with their ad libitum counterparts. Birth weight was not affected (P > 0.10) by grand dam size; however, lambs born to H-offspring grew faster from birth until d 49 of age (P = 0.03). In conclusion, dam nutrition during pregnancy affected the resulting milk production of the offspring and composition and growth of their lambs. In addition, dam size affected the milk production of the offspring, lactose yield, and growth of their lambs. These findings are important for furthering our understanding of how the environment to which the female fetus is exposed can affect her subsequent development and her ability to nourish the next generation.

Intrauterine environments and breast cancer risk: meta-analysis and systematic review

IntroductionVarious perinatal factors, including birth weight, birth order, maternal age, gestational age, twin status, and parental smoking, have been postulated to affect breast cancer risk in daughters by altering the hormonal environment of the developing fetal mammary glands. Despite ample biologic plausibility, epidemiologic studies to date have yielded conflicting results. We investigated the associations between perinatal factors and subsequent breast cancer risk through meta-analyses.MethodsWe reviewed breast cancer studies published from January 1966 to February 2007 that included data on birth weight, birth order, maternal age, gestational age, twin status, and maternal or paternal smoking. Meta-analyses using random effect models were employed to summarize the results.ResultsWe found that heavier birth weights were associated with increased breast cancer risk, with studies involving five categories of birth weight identifying odds ratios (ORs) of 1.24 (95% confidence interval [CI] 1.04 to 1.48) for 4,000 g or more and 1.15 (95% CI 1.04 to 1.26) for 3,500 g to 3,999 g, relative to a birth weight of 2,500 to 2,599 g. These studies provided no support for a J-shaped relationship of birthweight to risk. Support for an association with birthweight was also derived from studies based on three birth weight categories (OR 1.15 [95% CI 1.01 to 1.31] for ≥4,000 g relative to <3,000 g) and two birth weight categories (OR 1.09 [95% CI 1.02 to 1.18] for ≥3,000 g relative to <3,000 g). Women born to older mothers and twins were also at some increased risk, but the results were heterogeneous across studies and publication years. Birth order, prematurity, and maternal smoking were unrelated to breast cancer risk.ConclusionOur findings provide some support for the hypothesis that in utero exposures reflective of higher endogenous hormone levels could affect risk for development of breast cancer in adulthood.

Exposure to Environmentally Relevant Doses of the Xenoestrogen Bisphenol-A Alters Development of the Fetal Mouse Mammary Gland

Humans are routinely exposed to bisphenol-A (BPA), an estrogenic compound that leaches from dental materials, food and beverage containers, and other plastic consumer products. Effects of perinatal BPA exposure on the mouse mammary gland have been observed in puberty and adulthood, long after the period of exposure has ended. The aim of this study was to examine fetal mammary gland development at embryonic day (E)18 and assess changes in the tissue organization and histoarchitecture after exposure to an environmentally relevant dose of BPA. In unexposed fetuses, the relative position of the fetus with respect to its female and male siblings in the uterus influenced growth of the ductal tree, which was more developed in females placed between two males than in females placed between two females. Exposure of dams to 250 ng BPA per kilogram body weight per day from E8 to E18 significantly increased ductal area and ductal extension in exposed fetuses and obliterated positional differences. In the stroma, BPA exposure promoted maturation of the fat pad and altered the localization of collagen. Within the epithelium, BPA exposure led to a decrease in cell size and delayed lumen formation. Because mammary gland development is dependent on reciprocal interactions between these compartments, the advanced maturation of the fat pad and changes in the extracellular matrix may be responsible for the altered growth, cell size, and lumen formation observed in the epithelium. These results suggest that alterations in mammary gland phenotypes observed at puberty and adulthood in perinatally exposed mice have their origins in fetal development.

Adverse Effects of Prenatal Exposure to Atrazine During a Critical Period of Mammary Gland Growth

Prenatal exposure to 100 mg/kg atrazine (ATR) delays mammary gland (MG) development in resulting female offspring of Long-Evans rats. To determine if the fetal MG was sensitive to ATR effects during specific periods of development, timed-pregnant dams (n = 8/group/block) were dosed for 3- or 7-gestation day (GD) intervals (GD 13-15, 15-17, 17-19, or 13-19) with 100 mg ATR/kg/day or vehicle (C), and their offspring were evaluated for changes. Mammary glands taken from pups prenatally exposed to ATR displayed significant delays in epithelial development as early as postnatal day (PND) 4 compared to C, with continued developmental delays at later time points that varied by time of exposure. However, the most persistent and severe delays were seen in the GD 17-19 and GD 13-19 ATR exposure groups, demonstrating statistically similar growth retardation. Because MG developmental deficits persisted into adulthood, we hypothesized that ATR-exposed animals may have had difficulties nursing their young. Females exposed prenatally to either ATR (as defined) or C (n = 4 litters/group) were bred, and the resulting F(2) offspring from GD 17-19 and GD 13-19 exposure groups were significantly smaller in body weight (BW) than C. In a separate study, it was determined that ATR (25-100 mg/kg), delivered from GD 15-19, did not decrease fetal body weights on GD 20, even though the higher doses significantly decreased weight gain of the dosed dams. These data suggest that the consequences of brief ATR exposure during a critical period of fetal MG development (GD 17-19), are both delayed MG development of the offspring and inadequate nutritional support of F2 offspring, resulting in adverse effects on pup weight gain.

Breast Cancer Incidence in Women Prenatally Exposed to Maternal Cigarette Smoke

Clinical studies show that maternal cigarette smoking reduces pregnancy estrogen levels. Women prenatally exposed to maternal cigarette smoke may, therefore, have a lower breast cancer risk because the fetal mammary gland's exposure to maternal estrogen is decreased. Associations between prenatal maternal cigarette smoke exposure and breast cancer, however, have not been observed in previous case-control studies that relied on exposure assessment after the onset of cancer. At the start of this study, cigarette smoking history was obtained directly from the mother. The National Cooperative DES Adenosis project was a follow-up study of health outcomes in women prenatally exposed to diethylstilbestrol (DES). At the start of the study, women's mothers provided information about cigarette smoking habits during the time they were pregnant with the study participant. In the current study, the breast cancer rates are compared among 4031 women who were or were not prenatally exposed to maternal cigarette smoke. The resultant relative rate (RR) is adjusted for potential confounding by other breast cancer risk factors using Poisson regression modeling. Fetal exposure to maternal cigarette smoke appeared to be inversely associated with breast cancer incidence (RR = 0.49; 95% confidence interval [CI] = 0.24-1.03). The inverse association was more apparent among women whose mothers smoked 15 cigarettes or fewer per day than among daughters of heavier smokers. There were, however, too few cases to precisely estimate a possible dose-response relationship. These results support the hypothesis that in utero exposure to maternal cigarette smoke reduces breast cancer incidence.

Spatial and temporal expression of insulin-like growth factor-I, insulin-like growth factor-II and the insulin-like growth factor-I receptor in the sheep fetal mammary gland.

The mammary gland is an example of a tissue of epidermal origin that depends for the development of its characteristic morphology on underlying mesenchymal cells. The interaction between mesenchyme and epithelium appears to be mediated by polypeptide growth factors. In situ hybridization has been used to study, in the mammary gland of female sheep fetuses, the distribution of mRNA for the mammary mitogens, insulin-like growth factor (IGF)-I and IGF-II, and the IGF-I receptor, from 10 to 20 weeks of intrauterine life (term is approximately 22 weeks). At 10 weeks, secondary ducts had formed from the primary duct. By week 20, the gland had increased in volume and complexity, showing primitive lobules embedded in intralobular connective tissue disposed around main ducts. IGF-I and IGF-II mRNA were expressed in cells of the intralobular connective tissue underlying the epithelium, while the IGF-I receptor was expressed in epithelium. Quantitation by absorbance measurements showed that mRNA expression increased with pregnancy stage for IGF-I and IGF-II, but not significantly for the IGF-I receptor, and that IGF-II was more highly expressed than IGF-I. A role for the IGF system in mediating mesenchymal epithelial interactions in mammary development is indicated.

Expression of growth hormone receptor in the bovine mammary gland during prenatal development.

Growth hormone (GH) is known to play a key role in postnatal growth and differentiation. The role of GH and its receptor (GHR) in prenatal development, however, is still controversial. Using reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemistry we demonstrated the presence of GHR mRNA and protein in bovine mammary glands during fetal development. RT-PCR revealed GHR transcripts in fetal mammary glands from the third to the ninth month of pregnancy. By non-radioactive ISH, GHR mRNA was localized in the glandular epithelium, the surrounding mesenchymal cells, endothelial cells of vessels and in the stratum basale of the epidermis of fetal mammary glands. From the sixth month of fetal life onwards, GHR transcripts were also found in the cytoplasm of adipocytes. Immunohistochemical studies using the monoclonal antibody mAb 263 revealed the same distribution pattern as the mRNA. Our results imply that the growth hormone receptor is distinctly expressed in the immature mammary gland, suggesting that GH is involved in growth and differentiation of the fetal mammary gland.