Regulation of prostaglandin receptors and P450c17 by PGE2 during ovine pregnancy and parturition

Rationale: Preterm birth (<37 weeks gestation), which affects women in both developed and underdeveloped countries, occurs in about 10% of all pregnancies and accounts for approximately 70% of neonatal mortality and morbidity.  Individuals born prematurely are at an increased risk of developing respiratory and neurological problems in early childhood that can continue into adulthood.  Preterm birth imparts significant emotional stress for the affected family members and treatment of preterm infants has a major financial impact on the health care community.  By understanding the endocrine events involved in labour onset, new strategies and/or medical technologies can be developed to identify new markers of preterm birth and to prevent its occurrence. 

Background: Prostaglandins (PGs), such as PGE₂ and PGF_2α, have been well described as important effectors in the onset of parturition where they are known to play a key role in cervical ripening, uterine contractility, membrane rupture, and in the endocrine pathway through stimulation via the hypothalamic-pituitary-adrenal axis (HPA axis).  PGs mediate these effects through specific G-protein coupled receptors - the EP receptors (subtypes1-4) for PGE₂ and FP receptor for PGF_2α.  Stimulation of contractile receptors (EP1, EP3, FP) results in Ca²⁺ mobilization and cAMP inhibition, in contrast to relaxatory receptors (EP2, EP4) that promote cAMP production.  Previous studies in sheep reported that fetal cortisol produced in late gestation is associated with increased placental PGHS-2 (a prostaglandin synthase) protein expression, and a subsequent increase in PGE₂ synthesis. This rise in PGE₂ induces placental P450c17, an enzyme that converts C21 steroids to C19 sex steroid precursors, resulting in altered placental steroid synthesis at term.  Also, we previously demonstrated that periconceptional undernutrition (UN) results in a precocious rise in fetal cortisol levels and subsequent increases in PG concentrations and estrogen synthesis, resulting in preterm delivery.  However, the mechanism by which PGE₂ increases P450c17 is not understood.  

Objectives:  Using two different models, glucocorticoid-induced preterm labour and a periconceptional undernutrition model, we aim to investigate the role of PGs in altering placental steroid synthesis, in late pregnancy and parturition, by assessing whether PGE₂ acts as an intermediary through which increased fetal cortisol levels result in increased P450c17 expression and to determine the expression, localization and regulation of PG receptors that modulate this effect.    

Hypothesis: We propose that in late pregnancy and labour, placental PGE₂, acting through EP receptor subtypes 1-4, directly upregulates P450c17 in an autocrine/paracrine, and/or intracrine manner.  Differential regulation of the EP receptors in late gestation ovine placentomes might be affected by cortisol +/- meloxicam (PGHS-2 inhibitor) infusion and/or periconceptional UN.

Experimental design & methodology: A.) Glucocorticoid-induced preterm labour model: Late gestation (d120-125) pregnant ewes were chronically instrumented with maternal and fetal vascular catheters, amniotic fluid catheters, and uterine electromyogram (EMG) leads.  After ~5d recovery, ewes were randomly divided into 3 experimental groups: 1.) saline control (S; n=6); 2.) intra-fetal cortisol (C; n=4); 3.) intra-fetal cortisol + meloxicam (CM; n=5).  Animals were infused for an 80hr time period.  Uterine contractility (via EMG) and intrauterine pressure (IUP) were continuously recorded and blood gases were monitored daily to assess fetal and maternal health.  Blood samples were collected every 8 hrs beginning 24hrs prior to infusion start and over the 80hr time period.  At infusion end (~130d gestation; term=145d), ewes were anaesthetized and placentomes were rapidly dissected, frozen on dry ice and stored at 80⁰C until analysis. Placental protein expression levels of PGHS-2, EP1-3 subtypes and FP were analyzed by Western blot with corresponding preabsorption controls.  Plasma concentrations of cortisol and PGE₂ (fetal) and progesterone, estradiol, and 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM; metabolite of PGF_2α) (maternal) will be analyzed using radioimmunoassay (RIA) and ELISA.  B.) Periconceptional undernutrition model: Singleton-bearing Romney ewes were randomized to either control (ad libitum feed, N, n=7) or UN (n=10) from 60 days before conception until 30 days after, followed by maintenance feeds for the remainder of pregnancy in all ewes.  The UN regimen reduced maternal body weight by 15% and this recovered upon re-feeding.  At 131d gestation, ewes were euthanized and placentomes were rapidly dissected, frozen on dry ice and stored at -80⁰C until analysis. Placental protein expression levels of EP1-4 subtypes were analyzed by Western blot with corresponding preabsorption controls.  Immunohistochemical localization of EP1-4 was performed using antigen retrieval.

Results: A.) At the end of 80hrs (~130d gestation), PGHS-2 protein expression was significantly increased in both C and CM groups versus S control (p < 0.05).  Protein levels of EP1-3 and FP protein in placentomes were unaltered by C or CM infusion.  (Note: EP4 protein expression and all plasma measurements have yet to be completed).  B.) At 131d gestation, EP1-4 protein was expressed in both N and UN placentomes and protein levels were similar between the two groups.  All EP receptor subtypes were localized to the fetal trophoblast layer and endothelium of blood vessels and were also localized to glandular epithelium in full membrane sections.  Distribution of EP1-4 receptor subtypes were similar between N and UN groups, consistent with the protein data.

Conclusions: A.) The expression pattern of EP1-3 subtypes in late gestation ovine placentomes suggests that PGE₂, in late gestation, likely acts through EP receptors to regulate levels of P450c17.  We conclude that enhancement of PGE₂, through increased PGHS-2 enzyme, rather than alteration in prostaglandin receptor subtype expression, is responsible for the established pattern of altered placental steroid biosynthesis and parturition in this species.  B.) The expression pattern of EP1-4 subtypes in N and UN placentomes suggests that PGE₂, in late gestation, likely acts through EP receptors to regulate levels of P450c17.  Thus, EP receptors likely mediate this regulation in an autocrine/paracrine and/or intracrine manner.  Our data suggest that UN does not have a significant effect on the distribution and protein levels of EP receptors in ovine placentomes, consistent with previous studies from our lab demonstrating that UN did not affect placental P450c17 protein levels.

New knowledge gained and transdisciplinarity of research project:

With the recent birth of sextuplets in Vancouver, there has been much media coverage focused on reproductive technologies and premature birth.  A current CBC news article has highlighted the difficulties premature infants face when brought into the world "ahead of schedule".  Thus, the anticipated results of my research project will assist in understanding the mechanisms underlying term and preterm birth.  With the results that have been generated to date from my research, a better understanding of the placental endocrine axis of labour has been elucidated.  With this new knowledge, current tocolytic therapies can be modified or novel drugs and markers of preterm birth can be identified, which aim to improve the overall health and well-being of the mother and baby. 

Although my particular research project focuses on the basic sciences, it is important to address other transdisciplinary aspects with respect to preterm birth (PTB).  Economically, management of PTB comes at a significantly high cost to the health care system.  Caring for a premature infant can cost $1000-2000/day in hospital and particular tocolytic therapies may also prove to be costly depending on the individual case presented (i.e. administration of antenatal glucocorticosteroid treatment + tocolytic drug therapy).  Moreover, long-term follow-up studies are important in order to determine the potential effects of certain treatments on maternal health and fetal growth and development.  The social impact of PTB is also high as families are emotionally affected by having an infant who requires care in a neonatal intensive care unit (NICU).  Many parents and families who have had an infant in an NICU have experienced the sometimes day-to-day uncertainty of their child's health.  The time the infant is hospitalized not only brings about this large amount of stress, but some families might have to travel long distances in order to have their infant cared for in a tertiary care NICU.  Additionally, it can potentially bring financial burden to the family as both parents might have to cut back on the amount of time they spend working.  Continuing with another transdisciplinary theme, it is also important to recognize the ethical issues pertaining to preterm labour.  For example, the number of cases of multi-fetal pregnancies, which often result in PTB, continues to rise and many scientists and health care professionals cite that this is partly due to the increased use of IVF and other assisted reproductive technologies.  IVF and other infertility treatments continue to bring to light the issue of ethics.  Despite the fact that IVF is routinely used in infertility treatment, some individuals cannot draw a line between the patient and the research subject in these instances and, as a result, have a difficult time accepting them as routine clinical practice.  Additionally, there are some individuals who argue that it may not be in the best interest of the baby to prevent premature birth as the infant may initiate labour in order to remove itself from an adverse situation in utero.  Thus, overall it is important for health care professionals and researchers to consider all facets and complexities of PTB in order to optimize the health and well-being of the mother and infant.

Questions:

1. Although placental PGE₂ levels increase in late gestation, why do we not see an upregulation in EP receptors? Can you suggest an underlying mechanism?

2. In your opinion, do you agree/disagree when it comes to preventing or delaying premature birth?