Assisted reproduction technologies alter steroid delivery to the mouse fetus during pregnancy

Abstract

Assisted reproduction technologies (ART) include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), and are common treatments for infertility. Although generally successful, ART warrant further investigations due to emerging perinatal issues, especially low birth weight. Herein we extend our previous work demonstrating higher steroid clearance in murine ART placentas by examining steroid biosynthesis and the directional flow of steroids in the maternal–placental–fetal units. The activities of the major steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 17-αhydroxylase (CYP17) were assessed in maternal liver and ovaries and fetal livers as were levels of cholesterol, progesterone, estrone (E1), and estradiol (E2) in the maternal, placental and fetal units. No structural abnormalities were found in placentas from ART. Although ART increased 3β-HSD activity in maternal livers, there were no other changes in 3β-HSD- or CYP17-mediated steroidogenesis. Cholesterol levels were significantly lower in maternal livers of ICSI pregnancies and in placentas from both IVF and ICSI pregnancies but not altered in the fetal livers. Progesterone levels were higher in maternal and fetal livers in IVF and ICSI, respectively, but were significantly lowered in ICSI placentas, compared to normal fertilization. For estrogenic hormones, no differences in E1 or E2 levels were observed in maternal livers but ICSI significantly increased both E1 and E2 levels in placentas while both IVF and ICSI significantly lowered E1 but raised E2 levels in fetal livers. In summary, while steroid production was normal, steroid diffusion/flow from mother to fetus was altered in murine pregnancies conceived by ART. This appears to occur, at least in part; through placental mechanisms. Impaired cholesterol and steroid transfer may affect correct regulation of fetal growth and development.

Introduction

The use of assisted reproduction technologies (ART) is increasing dramatically in the developed world [1]. While ART is considered a relatively safe and effective way to conceive, in vitro fertilization (IVF) with or without intra-cytoplasmic sperm injection (ICSI), confers a higher risk of adverse reproductive outcomes compared to couples who conceive naturally. These adverse outcomes include higher incidences of induced labor, cesarean section, premature birth, small-for-gestational age babies, pediatric cancer, imprinting disorders and congenital abnormalities [2], [3], [4], [5], [6]. In addition to neonatal outcomes; higher rates of placenta previa, placental abruption, premature rupture of the membranes, pre-eclampsia, unusual placental shape and umbilical cord insertion in humans and higher placental weights in mice are known to occur with ART [5], [7], [8], [9], [10]. The etiology of such complications is unknown, but in addition to direct maternal causes, the placental and fetal origins of pregnancy outcomes should also be considered.

In human pregnancies, the major site of steroid production is the feto-placental unit, specifically the placenta, fetal adrenal and liver with the primary building-block (cholesterol) being the only major contribution from the maternal circulation. However, in mice pregnancies the maternal ovaries are the major site of sex steroid (estrone, estradiol, progesterone) production with a moderate contribution from placenta in the first half of gestation, and from the fetal tissues in the second half of gestation [11]. In both mice and humans, the production of these steroids from cholesterol is mediated upstream by the steroidogenic enzymes 3β hydroxysteroid dehydrogenase (3βHSD) and cytochrome P450 17α-hydroxylase (CYP17).

We have recently reported that functional changes in placental clearance of steroids occurred in mice when conception was achieved by ART [7] providing preliminary evidence that altered placental function may be responsible for some of the adverse reproductive outcomes reported for ART. Specifically we demonstrated bigger placental size in ART fetuses and dysregulation in steroid hormone metabolism and clearance across the placenta but no differences in maternal ovarian progesterone and estrogen levels in ART [7]. To expand our understanding of the mechanisms by which ART alters steroids, we wished to examine steroidogenesis and the directional flow of steroids in the maternal–placental–fetal units of normal and ART pregnancies. We generated a new cohort of ART and normal fertilization pregnancies, performed gross pathology and histological analyses on placental tissues, assessed activity of the steroidogenic enzymes 3βHSD and CYP17 in ovaries and maternal and fetal livers, and defined the levels of cholesterol, progesterone, estrone (E1) and 17β-estradiol (E2) in placentas and maternal and fetal livers. These tissues were chosen since they are the major site of production for estrone, estradiol, and progesterone during pregnancy as well as because of their major role in removing these steroids.

We demonstrated that IVF and ICSI do not alter placental structure nor do they alter ovarian or fetal liver steroid production by the enzymes 3βHSD and CYP17. However, we also showed that the net diffusion and/or transport of the steroids, progesterone, estrone and 17β estradiol, as well as their precursor cholesterol from the maternal circulation through the placenta to the fetus was altered in ART compared to normal pregnancies. This agrees with and strengthens our previous report showing higher steroid metabolism and clearance in the placenta [7] and further support our hypothesis that abnormal placental function may be responsible for adverse reproductive outcomes.