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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Dev Neurobiol. 2016 Oct 26;77(6):767–774. doi: 10.1002/dneu.22462

Progesterone from Maternal Circulation Binds to Progestin Receptors in Fetal Brain

Christine K Wagner 1, Princy Quadros-Mennella 2
PMCID: PMC8972071  NIHMSID: NIHMS1791596  PMID: 27739256

Abstract

Steroid hormones activate nuclear receptors which, as transcription factors, can regulate critical aspects of neural development. Many regions of the rat forebrain, midbrain and hindbrain express progestin receptors (PR) during perinatal life, suggesting that progesterone may play an important role in the development of the brain. An immunohistochemical approach using two antibodies with differential recognition of ligand-bound PR was used to examine whether fetuses are exposed to maternal progesterone during pregnancy and whether progesterone from maternal circulation can bind to PR within the fetal brain. Findings demonstrate that maternal and fetal serum progesterone levels are positively correlated at the end of gestation, suggesting a common source of progesterone in mothers and fetuses (e.g., the maternal ovary). Additional findings suggest that administration of exogenous progesterone to mothers not only increases fetal serum progesterone levels within 2 h, but appears to increase ligand-bound PR in fetal brain. These findings suggest that progesterone of maternal origin may play a previously overlooked role in neural development. In addition, there are implications for the ongoing prophylactic use of synthetic progestins in pregnant women for the prevention of premature birth.

Keywords: steroid receptors, medial preoptic nucleus, progesterone, antibodies, development

INTRODUCTION

Steroid hormones exert profound effects on the maturation of the nervous system through the activation of nuclear receptors which, as transcription factors, can influence fundamental mechanisms of neural development. This has been most notably demonstrated in the ability of the fetal gonadal hormone testosterone and its metabolites to produce marked and permanent sex differences in brain structure and function [for recent reviews see (Lenz et al., 2012; de Vries et al., 2014)]. However, it is becoming increasingly clear that the developing brain is also sensitive to progestins during critical periods of maturation (Quadros et al., 2007, 2008; Lopez and Wagner, 2009; Jahagirdar and Wagner, 2010) and that progesterone may play a previously underappreciated role in neural development.

Many regions of the developing rodent brain express nuclear receptors for progestins. Progestin receptor (PR) expression is transient in some regions, present only during late fetal and early postnatal life, and then absent throughout adulthood (Quadros et al., 2007, 2008; Lopez and Wagner, 2009; Jahagirdar and Wagner, 2010; Willing and Wagner, 2016). For example, PR is transiently expressed in dopaminergic areas of the midbrain, nuclei of the hindbrain and regions of the forebrain including neocortex and hippocampus. In some areas of the preoptic area and hypothalamus, PR is differentially expressed between the sexes (Wagner et al., 1998; Quadros et al., 2002a; Quadros et al., 2002c; Gonzales et al., 2008), suggesting that progesterone and its receptor may play a role in sexual differentiation (Lonstein et al., 2001; Quadros et al., 2002b). For example, in the medial preoptic nucleus (MPN), PR expression is high in males beginning around embryonic day 18 (E18), but is virtually absent in the female MPN until the second week of life (Wagner et al., 1998; Quadros et al., 2002a). Taken together, these findings suggest that progesterone may be integrally involved in numerous aspects of neural development important for both reproductive and cognitive behaviors later in life.

Within the last decade, the administration of natural progesterone (P) or the synthetic progestin, 17α-hydroxyprogesterone caproate (17-OHPC) to pregnant women for the prevention of premature birth has risen dramatically (Ness et al., 2006), with 17-OHPC receiving FDA approval in 2011. Pregnant women who are “at risk” for preterm delivery (i.e., have had a prior premature birth) typically receive 17-OHPC or natural progesterone (intramuscularly and/or vaginally) prophylactically beginning between weeks 16 and 20 of gestation and continuing through about week 36 (Berghella, 2012; O’Brien, 2012; Schmouder et al., 2013; Suhag et al., 2015). In vitro studies demonstrate that 17-OHPC (both the parent compound and its metabolites) can be transferred from the maternal circuit to the fetal circuit of human placenta (Hemauer et al., 2008), and can be detected in fetal plasma at least 44 days after the last injection, likely due to the fact that maternal fat stores serve as a repository (Caritis et al., 2012). This raises the possibility that progestins in maternal circulation may reach the fetal brain and influence neural development (Willing and Wagner, 2016)

Using the rodent MPN as a model, the present studies investigated whether progestins from maternal circulation can enter the fetal bloodstream, and moreover, reach the fetal brain and bind to PR. Traditional autoradiography studies are not definitive in this regard as it is virtually impossible to remove all competing endogenous progesterone and still maintain pregnancy viability and placental health. Therefore, we utilized an immunochemical approach and took advantage of two PR antibodies with differential recognition of ligand-bound PR. Using both antibodies, we assessed PR immunoreactivity in the MPN of male fetuses whose mothers had received a single bolus injection of progesterone or vehicle 2 h prior.

METHODS

Experimental Design

In Experiment 1, serum progesterone levels were measured in pregnant dams and their male and female fetuses at the end of pregnancy (i.e., E20, E21, E22) when maternal levels of progesterone fall prior to parturition.

For Experiment 2, pregnant dams received a single bolus injection of progesterone or the oil vehicle alone on the morning of E22 (Fig. 1). Two-hours later, blood was collected from dams and their male and female fetuses, and brains from fetal males and dams were collected for immunocytochemistry for PR, as fetal males alone demonstrate PR expression in the MPN (10). PRir was detected in the MPN of dams and their male fetuses using either an antisera that recognizes both liganded and unliganded PR (DAKO) or an antibody that preferentially recognizes unliganded PR (H928) to assess the degree to which progesterone in maternal circulation can bind to PR in the fetal brain.

Figure 1.

Figure 1

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Experimental Design Experiment 2: Pregnant females were injected with either progesterone (P) or the oil vehicle on embryonic day 22 (E22). Two-hours later, maternal and fetal blood was collected and serum progesterone levels determined by radioimmunoassay. Maternal and fetal male brains were collected and sections through the medial preoptic nucleus were processed for immunocytochemistry for progesterone receptor (PR) using a DAKO PR antisera which recognizes both ligand bound and unbound PR or, in sections from the same animals, using a H928 PR antisera which preferentially recognizes unbound PR. Decreased H928 PR immunoreactivity is taken as an indication of increased P binding to PR.

Animals.

All animal procedures were approved by the Institutional Animal Care and Use Committee at UMASS Amherst or University at Albany. Adult Sprague-Dawley female rats (Taconic Farms) were mated and the day a copulatory plug was found was designated as embryonic day 1 (E1). Pregnant females were maintained in a temperature and light controlled room (14:10 light:dark) with food and water available ad libitum.

Serum Progesterone Levels in Dams and Fetuses.

On the morning of E20, E21, and E22, pregnant females (N = 3) were anesthetized with sodium pentobarbital. The thorax and abdomen were opened and maternal blood was drawn by cardiac puncture, transferred to heparinized tubes and stored at −22°C. Meanwhile, fetuses were removed from the uterine horns and amniotic sacs and sacrificed by decapitation. Fetal trunk blood was collected into heparinized tubes. Fetuses were sexed by the detection of the SRY gene utilizing polymerase chain reaction as described previously (Quadros et al., 2002a).

Radioimmunoassay for Serum Progesterone.

Total progesterone was measured in maternal serum and male and female fetal serum using radioimmunoassay (125I RIA kit from MP Biomedicals). Twenty-five microliter of serum was added to 75 μL of diluent (phosphaline gelatin buffer (pH 7.0) containing rabbit gamma globulins), followed by 500 μL of anti-progesterone and 200 μL [125I]-progesterone. Tubes were incubated at 37°C for 60 min. Bound counts were precipitated by the addition of 500 μL of precipitant solution (phosphaline gelatin buffer and goat anti-rabbit gamma globulins). Tubes were then centrifuged at 3500 rpm (1000g) for 30 min. The supernatant was aspirated and the pellet counted on a gamma counter. Standards were calibrated to measure serum progesterone levels from 0.2 to 25.9 ng/mL. Correlations between maternal and fetal levels of serum progesterone were determined using Pearson Correlation Coefficient (p < 0.05).

Progesterone Administration on E22.

On the morning of E22 (0800–1000 h) pregnant females received a single injection of progesterone (8mg in 1.0 cc sesame oil, s.c.) or an equal volume of the oil vehicle alone. This dose was empirically determined to produce significantly elevated levels of progesterone at E22 but that did exceed the highest levels of endogenous progesterone observed during pregnancy (see Fig. 3 inset). Exactly 2 h following injection, pregnant females were administered a lethal dose of sodium pentobarbital. Maternal blood was drawn by cardiac puncture transferred to heparinized tubes and placed on ice. The dam was intracardially perfused with 0.9% saline followed by 5% acrolein (Sigma Aldrich, Inc) in 0.1M phosphate buffer (pH 7.4). Maternal brains were removed and post-fixed in 5% acrolein for 2 h. Fetuses were removed from the uterine horn and killed by rapid decapitation. Trunk blood from male and female fetuses was collected into heparinized tubes. Fetal sex was determined by the presence of testes or uterine horns. Fetal brains were removed and immersion fixed in 5% acrolein for 6 h. Serum progesterone levels of dams and fetuses were determined by radioimmunoassay as described above. Group differences were analyzed using Student t-tests (p < 0.05).

Figure 3.

Figure 3

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(A) Serum progesterone (P) levels in mothers that received an injection of progesterone or vehicle 2 h prior. Inset shows comparison of serum progesterone levels of P-injected and control dams on E22 to endogenous progesterone levels in pregnant female at E19. (B) Serum progesterone levels in the fetuses of mothers shown in (A). *significantly different from controls **p < 0.01; *p < 0.05.

Antibodies.

PR immunoreactivity was detected using two different immunological agents: the rabbit polyclonal antiserum (DAKO) (DAKO Corp., Glostrup, Denmark) (Traish and Wotiz, 1990; Lopez and Wagner, 2009) or the mouse monoclonal antibody (H928) (Stressgen, Biotechnologies Corp, Victoria BC, Canada) (Weigel et al., 1992; Park-Sarge et al., 1995).

DAKO:

DAKO polyclonal antiserum was raised against a synthetic antigen peptide corresponding to amino acids 533–547 near the DNA binding domain of the human PR which is contained in both A and B isoforms of the receptor (Traish and Wotiz, 1990). Work from our laboratory and others have demonstrated that nuclear DAKO immunoreactivity was completely eliminated in brain by preadsorption of the PR antiserum with either the antigen peptide or a tenfold molar excess of human PRA and PRB proteins or when the primary antiserum was omitted (Tetel et al., 1997; Quadros et al., 2002c). In addition, all immunoreactivity with the DAKO antiserum is abolished in transgenic mice that possess an insertional mutation in the PR gene (PR knock out mice) (Lopez and Wagner, 2009). The ability of the DAKO antiserum to recognize the epitope in the PR protein does not appear to be influenced by progestin binding (Auger et al., 2000) (and present results). In other words, DAKO recognizes both bound and unbound PR in brain.

H928:

H928 monoclonal antibody was raised to a synthetic antigen peptide corresponding to amino acids 523–536 in the hinge region of the chicken PR and is contained in both A and B isoforms of the receptor (Weigel et al., 1992). The ability of this antibody to recognize rat PR has been demonstrated in rat granulosa cells producing bands 115kD and 83kD on immunoblots, corresponding to the molecular weights of the A and B iosforms of PR, respectively (Weigel et al., 1989; Natraj and Richards, 1993; Park-Sarge et al., 1995). H928ir was eliminated in adult female brain following omission of the H928 primary antibody and following preadsorption of the H928 antibody with a PR rich extract (Auger et al., 1996). Previous studies have demonstrated that treatment of adult, estradiol-primed, ovariectomized females with progesterone decreased the number of H928-ir cells in several regions of the preoptic area and hypothalamus within 1 h but did not alter the number of DAKO-ir cells (Auger et al., 2000). Similarly, results from the present study demonstrate that H928ir is reduced in pregnant females within 2 h of a single injection of progesterone. It is highly unlikely that PR expression was down regulated by progesterone given the extremely short (1–2 h) time frame and no change in PRir in the very same brains as detected using DAKO antisera. The parsimonious conclusion is that progesterone binding to PR induces a conformational change in the PR protein that interferes with the binding of the H928 antibody to the epitope (Auger et al., 2000). Therefore, in the present study, relative levels of H928 immunoreactivity are taken as a reflection of progesterone binding to PR. Lower levels of H928-ir indicate higher levels of progesterone binding.

Immunocytochemistry for PR.

All brains were sectioned on a freezing microtome in the coronal plane at 50 μm. Alternate sections from each animal were collected and each set was processed for immunocytochemistry, one set for H928-ir and the other for DAKO-ir. Sections were stored in cryoprotectant (30% sucrose, 0.1% polyvinyl-pyrrolidone-40 in ethylene glycol, and 0.1 m phosphate buffer) at −20°C until immunocytochemical processing. Immunocytochemistry was performed on free floating sections using either the DAKO antisera or the H928 antibody described above. Sections were rinsed in tris-buffered saline (TBS; pH 7.6) three times for 5 min to remove any residual cryoprotectant solution. Sections were then incubated in 1% sodium borohydride in TBS for 10 min, rinsed in TBS four times for 5 min each, and then incubated in TBS containing 20% normal goat serum (NGS), 1% H2O2, and 1% bovine serum albumin for 30 min. DAKO PR antiserum was diluted to 1:1000 and H928 PR antibody was diluted 0.2 μg/mL in TBS containing 2% NGS, 0.3% Triton X-100 for 72 h at 4°C. Following three rinses (5 min each) in TBS containing 2% NGS, 0.3% Triton X-100, the sections were incubated for 60 min in biotinylated goat anti-rabbit IgG (for DAKO) or biotinylated goat anti-mouse IgG (for H928) (Vector Laboratories, Burlingame, CA) at a concentration of 5 μg/mL in TBS containing 2% NGS, 0.3% Triton X-100. After two rinses (5 min each) in TBS containing 2% NGS, 0.3% Triton X-100, and two rinses (5 min each) in TBS, the sections were incubated in avidin-biotin complex reagent (Vectastain Elite Kit, Vector Laboratories) for 60 min. Following three rinses (5 min each) in TBS, the sections were incubated in TBS containing 0.05% diaminobenzidine, 0.15% β-d-glucose, 0.04%ammonium chloride and 0.001% glucose oxidase for in TBS for 20 min. The sections were then rinsed three times (5 min each) in TBS and mounted on gelatin-coated slides and cover-slipped with Permount (Fisher Scientific, Pittsburgh, PA).

Image Analysis.

Anatomically matched sections through the MPN of each subject were selected for image analysis by an experimenter blind to treatment group. Microscopic images of the MPN were captured with a computer-assisted video camera (Diagnostic Instruments, Sterline Heights, MI) and NIH ImageJ was used to determine the mean optical density of PRir cells within the MPN. The mean optical density of an adjacent area devoid of PRir staining (i.e., background) was used to calculate the threshold value prior to analyzing the mean optical density of PRir cells. Group differences were analyzed using Student t-tests (p < 0.05).

RESULTS

Experiment 1

Maternal serum progesterone levels dropped precipitously at the end of pregnancy (i.e., between E20 and E22) (Fig. 1), as previously reported (Peppe and Rothchild, 1974; Sanyal, 1978). Serum progesterone levels in male and female fetuses, although lower than maternal levels, demonstrated a similar decrease between E20 and E22 (Fig. 1). Progesterone levels did not differ between male and female fetuses, therefore data from males and females were combined for correlational analysis. Maternal and fetal progesterone levels were significantly positively correlated (r = 0.8417, p < 0.008) (Fig. 1 inset).

Experiment 2

Serum progesterone levels were significantly higher in pregnant females that received a bolus injection of progesterone 2 h prior (p < 0.01) [Fig. 3(A)]. Although 8 mg is a relatively high dose of progesterone, resulting levels of progesterone in maternal circulation (~150 ng/mL) were still less than half of the levels of endogenous progesterone measured in pregnant females on E19 (~350 ng/mL) for comparison [Fig. 3(A) inset]. Serum progesterone levels did not differ between male and female fetuses (p > 0.05). Therefore, data from males and females were combined for statistical analysis. Serum progesterone levels in fetuses whose mothers received a progesterone injection were significantly higher than fetuses whose mothers received vehicle alone (p < 0.05) [Fig. 3(B)].

In the present study, relative levels of H928 immunoreactivity are taken as a reflection of progesterone binding to PR. Lower levels of H928-ir indicate higher levels of progesterone binding. The mean optical density of DAKO-ir in the MPN of mothers was not significantly different between progesterone-injected and control groups, suggesting that baseline levels of PR expression in the MPN were unaltered by progesterone in the 2 h following injection [Fig. 4(A)]. In contrast, the mean optical density of H928-ir in the same animals was significantly lower in pregnant females who received progesterone injections compared to controls (p < 0.005) [Fig. 4(B)].

Figure 4.

Figure 4

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Mean optical density of specific immunoreactivity detected with either (A) DAKO antisera (recognizing both ligand-bound and unbound PR) or (B) H928 antibody (preferentially recognizing ligand unbound PR) in the medial preoptic nucleus (MPN) of pregnant females who received a single injection of progesterone or oil vehicle 2 h prior. n.s. not significant; **p < 0.005.

A similar finding was observed in the MPN of male fetuses. There was no significant difference in levels of DAKO-ir in the MPN of males whose mothers received progesterone and those who received vehicle [Fig. 5(A)]. However, in the same animals, levels of H928-ir were significantly lower in males from P-injected mothers compared to controls (p < 0.01) [Fig. 5(b)].

Figure 5.

Figure 5

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Mean optical density of specific immunoreactivity detected with either (A) DAKO antisera (recognizing both liganded-bound and unbound PR) or (B) H928 antibody (preferentially recognizing unliganded PR) in the medial preoptic nucleus of male fetuses whose mothers received a single injection of progesterone or oil vehicle 2 h prior. n.s. not significant; **p < 0.01.

DISCUSSION

Maternal and fetal serum progesterone levels were positively correlated at the end of gestation in rats, suggesting that mothers and fetuses share a common source of progesterone and that fetuses may normally be exposed to progestins from maternal circulation. Furthermore, administration of progesterone to mothers significantly increased serum progesterone levels in fetuses compared to controls. An immunochemical approach demonstrated that progesterone from maternal circulation not only reaches fetal circulation, but binds to nuclear PR within fetal brain. PR, a powerful transcription factor, is expressed in many regions of the forebrain, midbrain and hindbrain during perinatal life (Quadros et al., 2007; Quadros et al., 2008; Lopez and Wagner, 2009). Taken together with the present results, this suggests that progesterone of maternal origin may play a significant role in fundamental mechanisms of fetal neural development.

In mammals, progesterone levels undergo rapid and extreme fluctuations during pregnancy (Peppe and Rothchild, 1974; Sanyal, 1978). In rats, peak levels of progesterone during gestation (~E19) are approximately ten times higher than at any other time during the life of the female (Butcher et al., 1974). Progesterone levels then decline precipitously, decreasing tenfold in just a few days (Sanyal, 1978). The primary source of progesterone during late gestation in rats is the maternal ovary, as the fetal female ovary is not yet steroidogenic (Quattropani and Weisz, 1973), and the precipitous decline in progesterone prior to parturition is due to the demise of the maternal corpus luteum (Sanyal, 1978). In the present study, maternal and fetal serum progesterone levels were positively correlated at the end of gestation. As maternal progesterone levels declined, fetal progesterone levels also declined, suggesting that the maternal ovary is the source of progesterone in fetal circulation. It is important to note that the relative contributions of maternal ovarian and placental progesterone secretion can vary across gestation and across species. For example, the placenta is the primary source of progesterone in later pregnancy in humans (Tuckey, 2005).

Administration of progesterone to pregnant female rats just prior to parturition, when endogenous progesterone levels are low, resulted in significantly elevated levels of progesterone in the serum of male and female fetuses within 2 h. Despite the relatively large dose of exogenous progesterone, resulting maternal serum levels were less than half that of levels seen just a few days earlier on day 19. These findings suggest that fetuses may be normally exposed to high and dynamic levels of endogenous maternal P, but may also be influenced by exogenous progestins in maternal circulation.

There were no sex differences in fetal serum progesterone levels, consistent with previous findings (Weisz, 1980) and with the fact that the fetal ovary is steroidogenically quiescent, not synthesizing the synthetic enzymes necessary for progesterone secretion until the second week of life in the rat (Quattropani and Weisz, 1973). However, sex differences in the expression of progesterone receptor in some brain regions may make male fetuses more sensitive to progesterone than females. For example, males express high levels of PR during perinatal life in several brain regions that regulate reproductive behaviors or neuroendocrine function later in life, whereas PR expression in females in these same regions is very low during development (Wagner et al., 1998; Quadros et al., 2002a; Quadros et al., 2002b). Therefore, progesterone and its receptor may play an important role in sexual differentiation despite similar circulating levels in males and females. Interestingly, there is a distinct absence of sex differences in PR expression in many other brain regions (e.g., hippocampus, cortex, dopaminergic midbrain nuclei) (Quadros et al., 2007, 2008; Lopez and Wagner, 2009), suggesting that males and females have a similar sensitivity to progesterone in these regions. This is consistent with the idea that progesterone may be critical in both sexes for the normal development of these brain regions many of which regulate behaviors such as cognition, learning, memory, and motivation. These findings generate the hypothesis that a complex interaction exits between sex, brain region and sensitivity to progesterone which permits similar levels of circulating progesterone to exert effects equally in males and females in some regions while still playing a role in the development of sex differences in other regions. This is in contrast to the well-described actions of testosterone in development, in which males and females are differentially exposed to testosterone secreted by the fetal testes regardless of the sensitivity of target tissues.

The present findings also suggest that progesterone from maternal circulation binds to PR in the fetal brain. The use of two antibodies with differential recognition of ligand-bound nuclear PR provided an indirect measure of progesterone binding in the maternal and fetal brains following exogenous progesterone administration. Fetuses whose mothers received progesterone had decreased levels of PRir detected with H928 whereas levels of PRir detected with DAKO were not altered within the same animals, consistent with the interpretation that progesterone from maternal circulation had bound to nuclear PR within the fetal MPN. While the possibility exists that H928ir was decreased as a result of the downregulation of PR expression by progesterone in the fetal brain, this explanation is unlikely for two reasons. First, the short 2 h survival time is unlikely to allow time for significant changes in transcription and translation of PR protein. Second, no decrease in DAKOir was observed in tissue from the same animals, suggesting that protein levels were similar between the groups. The more parsimonious interpretation is that progesterone binding to PR in fetal brain induced a conformational change in the PR protein which in turn masked the H928 epitope in the hinge region of the receptor, as previously suggested by Auger et al. (2000).

These findings also have implications for the rising use of progestins in pregnant women “at risk” for premature delivery. The most commonly prescribed synthetic progestin for this purpose, 17-OHPC, can cross the human placenta (Hemauer et al., 2008) and can be detected in fetal plasma (Caritis et al., 2012). The present results suggest that 17-OHPC may also reach the fetal brain. It remains unknown whether the human fetal brain expresses PR like the rodent brain. However, the possibility that this synthetic progestin could activate nuclear transcriptions factors within the fetal brain, perhaps to abnormal levels or at abnormal times compared to normal endogenous exposure, should be taken into consideration when evaluating outcomes. In fact, recent work from our laboratory has demonstrated that exposure to 17-OHPC during development in rats altered dopaminergic innervation of medial prefrontal cortex in pre-adolescence and impaired cognitive flexibility with increased perseveration in adulthood (Willing and Wagner, 2016).

In summary, the present findings suggest that progesterone from maternal circulation can enter fetal circulation and subsequently bind to nuclear receptors in fetal brain. In this manner, progesterone may play a critical role in normal brain development, and exposure to exogenous maternal progestins at inappropriate levels at inappropriate times may disrupt normal neural development. Careful consideration should be given to the use of synthetic progestins in pregnant women and our view of the steroid hormones that regulate brain development should not be limited to those secreted by the fetus.

Figure 2.

Figure 2

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Maternal and fetal serum progesterone (P) levels (ng/mL) on embryonic days 20 (E20) to E22. Inset: Serum progesterone levels in fetuses from individual litters plotted as a function of serum progesterone levels from individual mothers with regression line. Significant correlation (p < 0.008).

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