UC Berkeley

Osteoporosis is one of the most important health conditions affecting aging humans (≥ 60 years in age), particularly women in North America and Europe (Jordan and Cooper, 2002). One of the risk factors for osteoporosis is not attaining the maximum peak bone mass density for which an individual has the genetic potential. This risk factor is ascribed to poor environmental conditions and is irreversible (Cooper et al., 2009). Individuals who do not attain peak bone mass are at a significant disadvantage when aging sets in, with its accompanying loss of bone mass density. This places them at a higher risk for osteoporosis.

Psychological stress has been demonstrated to predispose pre-menopausal women to osteoporosis (Eskandari et al., 2007). Considering that environmental factors acting in pre-natal life have been known to influence adult health (Barker, 1995a, b, c, d; Gluckman et al., 2005), I hypothesized that psychological stress during pregnancy could result in high levels of cortisol that would affect bone formation in the fetus. This would compromise early bone development of the fetus and diminish the potential for attaining peak bone mass density in young adults, and therefore be associated with a higher risk for osteoporosis later in life.

To test this hypothesis I induced immobilization stress to pregnant Wistar rats at different gestational stages: Group 1 mothers were stressed during gestation week 1 (GW1), Group 2 during gestation week 2 (GW2), Group 3 during gestation week 3 (GW3); the Control Group was not stressed in any week. During gestation I monitored dams' cortisol hormone levels through fecal sampling, food intake, and maternal weight gain. After birth the pups were raised in a stress-free environment with adequate access to food and water and minimal human handling. Different sets of pups were euthanized at 4, 8, 12 and 16 weeks old. At necropsy the tibia was removed and fixed in 10% phosphate buffered formalin at 4°Celsius for 24 hours. The proximal part of the tibia (1 cm from the proximal end) was dehydrated in graduated series of ethanol and embedded in methyl methacrylate. Longitudinal sections, 4-μm thick were obtained using Leica 2165 Microtome (Leica, Heidelberg, Germany) with a tungsten carbide knife and placed on 2% gelatinized slides. The sections were stained using Von Kossa method with McNeal's tetrachrome counterstain. Bone histomorphometry was performed using semi-automated image analysis (Bioquant Image Analysis Corporation, Nashville, TN, USA) linked to a microscope to assess the size of the growth plate, trabecular total tissue area, trabecular bone area, trabecular bone perimeter, osteoblast surface, osteoid surface, erosion surface and number of osteoclasts.

The means and standard deviations were calculated for all outcome variables. Statistical differences between the stressed groups and control group were analyzed using t-test, F-test and Tukey-Kramer Honestly Significant Difference test (JMP. 2008. Version 8. SAS Institute Inc., Cary, NC). Linear regression analysis was performed to establish the relationship between stress in utero and indicators of bone development in offspring born to stressed mothers (StataCoRP. 2009. Stata; Release 11. Statistical Software. College Station, TX: StataCorp LP). P values equal to or less than 0.05 were considered significant.

The mean cortisol hormone levels in controls were consistently lower than those of all stressed groups. However, cortisol levels in the control group were found to increase over the duration of the pregnancy. The animals stressed in gestation week 1 had the highest cortisol hormone levels and were significantly different from controls during gestation week 1 (GW1 = 3.72μg/g, controls = 1.66μg/g, difference = 2.06μg/g), followed by those stressed in gestation week 2 (GW2 = 3.34μg/g, controls during GW2 = 1.73μg/g, difference = 1.62μg/g) and those stressed in gestation week 3 (GW3 = 3.36μg/g, GW3 controls = 2.38μg/g, difference = 0.98μg/g).

During the pregnancy period, stressed animals consumed 3 grams (12.5%) less food per day compared to the controls. It was noted that on the day before delivery, all the animals (stressed and controls) increased their food intake, almost doubling their norm.

The pregnant dams that were stressed during weeks 1 and 2 of their pregnancies gained significantly less weight over the duration of the pregnancy (GWI = 263.03 grams, GW2 = 277.64 grams) than did those stressed in week 3 or in the control group (GW3 = 315.40 grams, controls = 311.46 grams). The average number of pups born to females stressed in weeks 1 and 2 was greater (13 and 14 respectively) than for the controls or those stressed in week 3 (11 and 12 respectively). Both male and female offspring born to mothers stressed in GW3 were heavier compared to all the other groups, but the weight difference was not statistically significant.

Histological analysis was done on the offspring born to the dams stressed in gestation week 3 and the control group only. The decision to initially focus on GW3 offspring was based on the fact that this is the week during which rats' bones mineralize. Data collection for the histology phase of the project was very time-intensive. As such, the other experimental groups will be studied at a later date and the results reported elsewhere.

Histological analysis showed that males have larger bones compared to females starting at the age of 8 weeks for both offspring groups. Controlling for sex, there was no significant difference in trabecular total tissue area or the trabecular bone perimeter between the GW3 offspring and control offspring. The GW3 offspring had a higher bone formation rate as indicated by their higher trabecular bone area at the age of 8 weeks (GW3 = 2.16mm², controls = 1.27mm²), higher number of osteoblasts, which are the bone forming cells, at the age of 12 weeks (GW3 = 21.66mm, controls = 12.14mm) and a bigger area of the osteoid surface, which is the collagen matrix laid down by osteoblasts that eventually calcifies to form the bone, at the age of 8 weeks (GW3 = 4.98mm, controls = 1.92mm) and 12 weeks (GW3 = 5.50mm, controls = 0.99mm). There was no significant difference in bone resorption rate between the two groups.

The control offspring had a thicker upper zone of the growth plate, consisting of resting and proliferative chondrocytes, at the age of 4 weeks (GW3 = 302.83mcm, controls = 210.29mcm) and 8 weeks (GW3 = 195.73mcm, controls = 123.56mcm).

This project confirms that stress during pregnancy has negative consequences on both the mother and the offspring. The caloric intake in the mother is reduced, potentially due to the excess cortisol that alters the hypothalamic control of food intake. As a result, the mother does not accrue as many nutrients to support the growing fetuses. Having been nutrient-restricted and exposed to high cortisol levels in utero, the offspring appear to be born with an altered metabolism that results in faster growth and higher weight gain compared to controls. The positive effect of this fast growth is that the offspring born to stressed mothers ended up with a higher bone volume compared to the control offspring.

This study also shows that exposure to high cortisol levels in utero negatively affects the growth plate in offspring. Growth plate analysis showed that at the age of 4 weeks, control offspring had a significantly thicker area of resting and proliferative chondrocytes in the growth plate compared to GW3 offspring. Therefore, the negative effect of prenatal stress was evident in the upper zone of the growth plate even at the age of 4 weeks when the seemingly catch-up growth is expected to have occurred in all measured aspects of bone development. This seems to be the most sensitive part of bone development in relation to prenatal cortisol exposure.

In conclusion, given the negative effects of prenatal stress on the mother and offspring as noted above, this research shows that osteoporosis may have some fetal-origin roots influenced by maternal stress (and elevated cortisol levels). Healthy bones in adulthood require a healthy start. The growth plate is the center for bone growth and any adverse effects during early development would eventually affect the entire skeletal development. A likely result of not attaining the maximum peak bone mass density for which an individual has the genetic potential is a higher risk for osteoporosis.