Reproductive experience promotes permanent body growth independently of growth hormone

Reviewer #1 (Public review):

This work evaluates the impact of reproductive history on growth, body weight and body composition in mammals. In mice, somatic growth is stimulated by the first pregnancy while the second pregnancy increases body weight mainly by increasing adiposity. To probe the role of pituitary growth hormone (GH), the key regulator of somatic growth in these processes, was addressed by comparing the impact of reproduction on growth in normal ("wild type") and genetically GH-deficient females and by detailed characterization of the profile of fluctuations in circulating GH levels in both types of animals. Additional studies addressed the possible role of other endocrine pathways (ghrelin and estrogen) in the pregnancy-related growth. Surprisingly, reproduction-related growth was independent of GH, ghrelin and estrogen. To determine whether these results may apply ("translate") to human physiology, data on various parameters of somatic growth were collected from women with hereditary GH deficiency. The findings indicate that GH-independent stimulation of growth by reproductive events also occurs in women.

Use of multiple animal models, rigorous characterization of GH levels in normal and GH-deficient females, and inclusion of data derived from a unique and well-characterised population of people with hereditary isolated GH deficiency and no GH replacement therapy are important strengths of these elegant and innovative studies. The results address a broader and clinically significant issue of permanent changes in body size, composition and function that result from pregnancy and lactation. This work also provides important background for further studies aimed at the identification of the mechanism involved and the role of specific reproductive events in the regulation of growth.

Reviewer #2 (Public review):

This manuscript describes the fascinating phenomenon of growth hormone (GH)-independent growth occurring in the mother during pregnancy. This growth was most pronounced in dwarf mice that are lacking the receptor for growth hormone-releasing hormone (GHRH) and therefore showing isolated GH deficiency. However, the pregnancy-induced growth could also be observed in wild-type mice, suggesting that it is a normal part of the maternal adaptation to pregnancy. The study falls short of identifying the mechanism(s) driving this pregnancy-induced growth response, but it certainly reveals a novel insight into maternal physiology. The authors have completed a range of experiments in mice to prove that, as well as being GH independent, the pregnancy-induced growth also did not require GH signaling in the liver (i.e. not another pregnancy-specific ligand operating through the GHR to promote IGF). They also provided complementary data from a population of humans with untreated isolated GH deficiency that are broadly consistent with the hypothesis. While it is important to consider the significant species differences between rodents and humans, both in terms of growth physiology and also in terms of evolution of placental somato-mammotrophic hormones, this unique population are a valuable resource and adds credence to the study. Overall, I find this a compelling research story, but disappointingly unfinished. There are some areas where additional information could improve the ability to interpret the data, and some additional concepts that could be considered in the discussion. There are also areas where additional experiments might provide important insights. However, I think that such suggestions can be considered as appropriate for future research, rather than delaying consideration of the current manuscript.

Main comments:

(1) Data in Figure 1 are remarkable - not so much the growth in pregnancy in the wildtype mice, because while elevated GH is well known in pregnancy, but growth in the dwarf mice is indicative of GH-independent growth. From these data, it seems that there is good evidence that growth in pregnancy is an adaptive function. However, it is possible that growth is achieved in dwarf mice and that in wildtype mice may have been mediated through different mechanisms. The dwarf mice showed an increase in liver and plasma IGF1, suggestive of an additional ligand driving IGF in pregnancy. One could hypothesize that such an effect could be mediated by an additional pregnancy-specific ligand activating the GH receptor. In humans, placental growth hormone could be such a ligand, but as far as we know, there is no placental GH in mice. In contrast, the wildtype animals showed suppression of liver and circulating IGF1, and low levels of pSTAT5 in the liver during pregnancy. These data (in Figure 5) are very surprising. Given the high circulating GH in pregnancy, as well as high placental lactogen (which would be expected to activate STAT5 in the liver through the Prlr), the low levels of pSTAT5 are unexpected and would seem to indicate some sort of acquired insensitivity to GH. Is this entirely driven by down-regulation of STAT5b protein, or could there be activation of other, negative regulators of STAT signalling, such as SOCS? What is causing such a profound suppression of STAT5? Regardless of the mechanism, this suggests that pregnancy-induced growth in wildtype mice is independent of circulating IGF1 (potentially a different mechanism or in addition to that seen in IGHD mice).

The data shown in Figure 6 are a major strength of the study, showing that the pregnancy-induced changes are not specific to one particular transgenic model, but still occur in a variety of models affecting GH through different approaches. Given the pregnancy-specific nature of the changes, however, it seems an oversight not to have evaluated the role of placental lactogens. Prlr is highly expressed in the liver, but the function of this hormone in the liver is not well established. Could the extremely high levels of PL be mediating this growth response? Given the low expression of STAT5 in the liver and the fact that plasma IGF1 is not markedly elevated, it seems more likely that this growth response may be mediated by locally produced IGF1 in target tissues.

I think these possibilities could be addressed by an expanded discussion of species variation in placental hormones, to highlight that humans have expansion of the GH locus, but rodents have expansion of the prolactin axis (see Soares, M. J. The prolactin and growth hormone families: pregnancy-specific hormones/cytokines at the maternal-fetal interface. Reprod Biol Endocrinol 2, 51, 2004). Importantly, placental GH and chorionic somatomammotropins (CSM) in humans are all variants of the GH gene, but CSM have preferential activity at Prlr. This seems to be a fundamental species difference in pregnancy biology, but has been interpreted as an example of convergent evolution, with conservation of prolactin and GH-like functions at the maternal-fetal interface, mediated by different mechanisms, likely contributing to the metabolic adaptations of the mother (see Newbern D, Freemark M. Placental hormones and the control of maternal metabolism and fetal growth. Curr Opin Endocrinol Diabetes Obes. 2011; 18: 409-416). While the preceding function has focused on explaining the evolution of placental lactogens (either prolactin or GH variants), the present data suggest that there are also mechanisms to maintain growth in pregnancy, independent of GH (even in the absence of a placental GH).

(2) The human data are very interesting, and my initial impression was that it seemed unlikely to be the same phenomenon. Was there any real evidence for "growth" in pregnancy? Pubertal maturation of long bone growth might be expected to prevent further growth in adulthood. However, these issues were appropriately discussed, and it seems well justified to evaluate this unique population of women with IGHD who underwent pregnancy. It would be very interesting to know if these women experienced elevated IGF1 during pregnancy, indicative of placental GH contributing to growth. Mechanistically, this might be more like the dwarf mouse situation of IGHD, that the situation in wildtype mice (associated with liver insensitivity to GH and low IGF1).

(3) It would be useful to include investigations that isolate the effects of pregnancy and the placental hormones. Such studies could include evaluating growth in pseudopregnant mice with IGHD (pregnancy-like changes in hormones but lacking the placental contribution) and in IGHD animals that experience pregnancy but not lactation (pups removed at birth). I accept that this might be too large an additional study to add for the present manuscript.

(4) It is an important and translationally relevant observation that pregnancy increased the risk of long-term weight gain, and that after the first pregnancy, the pregnancy-induced growth response was more directed to promoting fat deposition. Does this provide any mechanistic insight? Could a metabolic adaptation result in growth?

Reviewer #3 (Public review):

Summary:

The study describes an increase in body growth and body composition in both mice and women. In mice, the impact on growth is mainly seen during the first pregnancy, and the changes postpartum on body composition are also different during the first and second pregnancies. The study has used various knock-out models in the growth hormone axis to understand these changes as well as some gene expression analysis related to GH, IGF-1 and estrogen signalling pathways.

Strengths:

(1) The inclusion of various knock-out mouse models that allow for exploration of mechanisms related to the above-mentioned changes.

(2) The investigation of gene expression of GHR, IGF-1R and ER pathways.

Weaknesses:

The human findings are dependent on the patient's recollection of bodily changes after their pregnancies.

Conclusion:

The authors have partly achieved their aim of describing changes in growth and body composition that remain after pregnancy and the mechanisms behind these changes. This study may have importance for a wide variety of research areas as well as in the clinical setting. The study is also unique in its attempt to bridge findings in mice to a unique human model of congenital GH deficiency.