IFNs Drive Development of Novel IL-15-Responsive Macrophages.

Disruption in homeostasis of IL-15 is linked to poor maternal and fetal outcomes during pregnancy. The only cells described to respond to IL-15 at the early maternal-fetal interface have been NK cells. We now show a novel population of macrophages, evident in several organs but enriched in the uterus of mice and humans, expressing the ß-chain of the IL-15R complex (CD122) and responding to IL-15. CD122+ macrophages (CD122+Macs) are morphologic, phenotypic, and transcriptomic macrophages that can derive from bone marrow monocytes. CD122+Macs develop in the uterus and placenta with kinetics that mirror IFN activity at the maternal-fetal interface. M-CSF permits macrophages to express CD122, and IFNs are sufficient to drive expression of CD122 on macrophages. Neither type I nor type II IFNs are required to generate CD122+Macs, however. In response to IL-15, CD122+Macs activate the ERK signaling cascade and enhance production of proinflammatory cytokines after stimulation with the TLR9 agonist CpG. Finally, we provide evidence of human cells that phenocopy murine CD122+Macs in secretory phase endometrium during the implantation window and in first-trimester uterine decidua. Our data support a model wherein IFNs local to the maternal-fetal interface direct novel IL-15-responsive macrophages with the potential to mediate IL-15 signals critical for optimal outcomes of pregnancy.

Superovulation with human chorionic gonadotropin (hCG) trigger and gonadotropin releasing hormone agonist (GnRHa) trigger differentially alter essential angiogenic factors in the endometrium in a mouse ART model†.

Gonadotropin-releasing hormone agonists (GnRHa) are used as an alternative to human chorionic gonadotropin (hCG) to trigger ovulation and decrease the risk of ovarian hyperstimulation syndrome. GnRHa is less potent at inducing ovarian vascular endothelial growth factor (VEGF), but may also affect endometrial angiogenesis and early placental development. In this study, we explore the effect of superovulation on endometrial angiogenesis during critical periods of gestation in a mouse model. We assigned female mice to three groups: natural mating or mating following injection with equine chorionic gonadotropin and trigger with GnRHa or hCG trigger. Females were killed prior to implantation (E3.5), post-implantation (E7.5), and at midgestation (E10.5), and maternal serum, uterus, and ovaries were collected. During peri-implantation, endometrial Vegfr1 and Vegfr2 mRNA were significantly increased in the GnRHa trigger group (P < 0.02) relative to the hCG group. Vegfr1 is highly expressed in the endometrial lining and secretory glands immediately prior to implantation. At E7.5, the ectoplacental cone expression of Vegfa and its receptor, Vegfr2, was significantly higher in the hCG trigger group compared to the GnRHa group (P < 0.05). Soluble VEGFR1 and free VEGFA were much higher in the serum of mice exposed to the hCG trigger compared to GnRHa group. At midgestation, there was significantly more local Vegfa expression in the placenta of mice triggered with hCG. GnRHa and hCG triggers differentially disrupt the endometrial expression of key angiogenic factors during critical periods of mouse gestation. These results may have significant implications for placental development and neonatal outcomes following human in vitro fertilization.

In Vitro fertilization and adverse obstetric and perinatal outcomes.

Most IVF-conceived children are healthy, but IVF has also been associated with adverse obstetric and perinatal outcomes as well as congenital anomalies. There is also literature suggesting an association between IVF and neurodevelopmental disorders as well as potentially long-term metabolic outcomes. The main driver for adverse outcomes is the higher risk of multiple gestations in IVF, but as the field moves toward single embryo transfer, the rate of multiple gestations is decreasing. Studies have shown that singleton IVF pregnancies still have a higher incidence of adverse outcomes compared to unassisted singleton pregnancies. Infertility itself may be an independent risk factor. Animal models suggest that epigenetic changes in genes involved in growth and development are altered in IVF during the hormonal stimulation and embryo culture. Further animal research and prospective human data are needed to elucidate the mechanisms by which IVF may contribute to adverse outcomes and to decrease risks.

miR-200 Regulates Endometrial Development During Early Pregnancy.

For successful embryo implantation, endometrial stromal cells must undergo functional and morphological changes, referred to as decidualization. However, the molecular mechanisms that regulate implantation and decidualization are not well defined. Here we demonstrate that the estradiol- and progesterone-regulated microRNA (miR)-200 family was markedly down-regulated in mouse endometrial stromal cells prior to implantation, whereas zinc finger E-box binding homeobox-1 and -2 and other known and predicted targets were up-regulated. Conversely, miR-200 was up-regulated during in vitro decidualization of human endometrial stromal cells. Knockdown of miR-200 negatively affected decidualization and prevented the mesenchymal-epithelial transition-like changes that accompanied decidual differentiation. Notably, superovulation of mice and humans altered miR-200 expression. Our findings suggest that hormonal alterations that accompany superovulation may negatively impact endometrial development and decidualization by causing aberrant miR-200 expression.

A Molecular Perspective on Procedures and Outcomes with Assisted Reproductive Technologies.

The emerging association of assisted reproductive technologies with adverse perinatal outcomes has prompted the in-depth examination of clinical and laboratory protocols and procedures and their possible effects on epigenetic regulatory mechanism(s). The application of various approaches to study epigenetic regulation to problems in reproductive medicine has the potential to identify relative risk indicators for particular conditions, diagnostic biomarkers of disease state, and prognostic indicators of outcome. Moreover, when applied genome-wide, these techniques are likely to find novel pathways of disease pathogenesis and identify new targets for intervention. The analysis of DNA methylation, histone modifications, transcription factors, enhancer binding and other chromatin proteins, DNase-hypersensitivity and, micro- and other noncoding RNAs all provide overlapping and often complementary snapshots of chromatin structure and resultant "gene activity." In terms of clinical application, the predictive power and utility of epigenetic information will depend on the power of individual techniques to discriminate normal levels of interindividual variation from variation linked to a disease state. At present, quantitative analysis of DNA methylation at multiple loci seems likely to hold the greatest promise for achieving the level of precision, reproducibility, and throughput demanded in a clinical setting.

The cumulative effect of assisted reproduction procedures on placental development and epigenetic perturbations in a mouse model.

Assisted reproductive technologies (ART) are associated with several complications including low birth weight, abnormal placentation and increased risk for rare imprinting disorders. Indeed, experimental studies demonstrate ART procedures independent of existing infertility induce epigenetic perturbations in the embryo and extraembryonic tissues. To test the hypothesis that these epigenetic perturbations persist and result in adverse outcomes at term, we assessed placental morphology and methylation profiles in E18.5 mouse concepti generated by in vitro fertilization (IVF) in two different genetic backgrounds. We also examined embryo transfer (ET) and superovulation procedures to ascertain if they contribute to developmental and epigenetic effects. Increased placental weight and reduced fetal-to-placental weight ratio were observed in all ART groups when compared with naturally conceived controls, demonstrating that non-surgical embryo transfer alone can impact placental development. Furthermore, superovulation further induced overgrowth of the placental junctional zone. Embryo transfer and superovulation defects were limited to these morphological changes, as we did not observe any differences in epigenetic profiles. IVF placentae, however, displayed hypomethylation of imprinting control regions of select imprinted genes and a global reduction in DNA methylation levels. Although we did not detect significant differences in DNA methylation in fetal brain or liver samples, rare IVF concepti displayed very low methylation and abnormal gene expression from the normally repressed allele. Our findings suggest that individual ART procedures cumulatively increase placental morphological abnormalities and epigenetic perturbations, potentially causing adverse neonatal and long-term health outcomes in offspring.

Pre-gestational vs gestational exposure to maternal obesity differentially programs the offspring in mice.

AIMS/HYPOTHESIS: Maternal obesity is associated with an increased risk of obesity and impaired glucose homeostasis in offspring. However, it is not known whether a gestational or pre-gestational exposure confers similar risks, and if so, what the underlying mechanisms are. METHODS: We used reciprocal two-cell embryo transfers between mice fed either a control or high-fat diet (HFD) starting at the time of weaning. Gene expression in placenta was assessed by microarray analyses. RESULTS: A pre-gestational exposure to a maternal HFD (HFD/control) impaired fetal and placental growth despite a normal gestational milieu. Expression of imprinted genes and genes regulating vasculogenesis and lipid metabolism was markedly altered in placenta of HFD/control. An exposure to an HFD (control/HFD) only during gestation also resulted in fetal growth restriction and decreased placental weight. Interestingly, only a gestational exposure to an HFD (control/HFD) resulted in obesity and impaired glucose tolerance in adulthood. CONCLUSIONS/INTERPRETATION: An HFD during pregnancy has profound consequences for the offspring later in life. Our data demonstrate that the mechanism underlying this phenomenon is not related to placental dysfunction, intrauterine growth restriction or postnatal weight gain, but rather an inability of the progeny to adapt to the abnormal gestational milieu of an HFD. Thus, the ability to adapt to an adverse intrauterine environment is conferred prior to pregnancy and it is possible that the effects of a maternal HFD may be transmitted to subsequent generations.

Peri-implantation hormonal milieu: elucidating mechanisms of abnormal placentation and fetal growth.

Assisted reproductive technologies (ART) have been associated with several adverse perinatal outcomes involving placentation and fetal growth. It is critical to examine each intervention individually in order to assess its relationship to the described adverse perinatal outcomes. One intervention ubiquitously used in ART is superovulation with gonadotropins. Superovulation results in significant changes in the hormonal milieu, which persist during the peri-implantation and early placentation periods. Epidemiologic evidence suggests that the treatment-induced peri-implantation maternal environment plays a critical role in perinatal outcomes. In this study, using the mouse model, we have isolated the exposure to the peri-implantation period, and we examine the effect of superovulation on placentation and fetal growth. We report that the nonphysiologic peri-implantation maternal hormonal environment resulting from gonadotropin stimulation appears to have a direct effect on fetal growth, trophoblast differentiation, and gene expression. This appears to be mediated, at least in part, through trophoblast expansion and invasion. Although the specific molecular and cellular mechanism(s) leading to these observations remain to be elucidated, identifying this modifiable risk factor will not only allow us to improve perinatal outcomes with ART, but help us understand the pathophysiology contributing to these outcomes.

Response to ovarian stimulation in patients facing gonadotoxic therapy.

Chemotherapy naïve patients undergoing embryo/oocyte banking for fertility preservation (FP) were assessed for response to ovarian stimulation. Fifty FP patients facing gonadotoxic therapy were matched by age, race, cycle number, date of stimulation and fertilization method to patients undergoing IVF for infertility or oocyte donation. There were no differences in baseline FSH, anti-Müllerian hormone, antral follicle count and total gonadotrophin dose. FP patients had more immature oocytes (2.2 versus 1.1; P=0.03) and lower fertilization rates per oocyte retrieved (52% versus 70%; P=0.002). There were no differences in numbers of oocytes retrieved, mature oocytes or fertilized embryos. Subgroup analysis revealed that FP patients taking letrozole required higher gonadotrophin doses (3077IU versus 2259IU; P=0.0477) and had more immature oocytes (3.4 versus 1.2; P=0.03) than matched controls. There were no differences in gonadotrophin dose or oocyte immaturity among FP patients not taking letrozole. Overall, chemotherapy naïve FP patients had similar ovarian reserve, response to stimulation and oocyte and embryo yield compared to controls. Patients who received letrozole required higher gonadotrophin doses and produced more immature oocytes, suggesting that response to ovarian stimulation may be impaired in patients with hormone-sensitive cancers receiving letrozole. With improvement in cancer survival rates, there has been a shift in attention toward management of long-term consequences of cancer therapy, including infertility. Many young women with cancer, particularly those who will be treated with chemotherapy, pursue fertility preservation (FP) strategies for the purpose of banking oocytes or embryos for future use. We examined patients with no prior exposure to chemotherapy who underwent IVF to freeze embryos or oocytes for FP. Fifty FP patients were identified and matched to healthy controls by age, race, cycle number, date of stimulation and fertilization method. There were no differences in baseline measures of ovarian reserve or amount of medication needed to stimulate the ovaries. FP patients had more immature oocytes and lower fertilization rates than controls. There were no differences in number of oocytes retrieved, number of mature oocytes, rate of maturity or number of fertilized embryos. Subgroup analysis revealed that FP patients taking letrozole required higher gonadotrophin doses and had more immature oocytes compared with matched controls. There were no differences in gonadotrophin dose or oocyte immaturity among FP patients not taking letrozole. We demonstrated that FP patients not previously exposed to chemotherapy have similar ovarian reserve, response to stimulation and oocyte and embryo yield compared with infertile and donor controls. Patients who received letrozole required higher gonadotrophin doses and produced more immature oocytes, suggesting that response to ovarian stimulation may be impaired in patients with hormone-sensitive cancers receiving letrozole.

Meiotic and developmental competence in mice are compromised following follicle development in vitro using an alginate-based culture system.

Culture systems that support development and maturation of oocytes in vitro with a high efficiency would have great impact not only on research addressed at underlying mechanisms of oocyte development but also on preservation of fertility. Recently, attention has turned to using culture systems that preserve follicle integrity, in contrast to existing systems that do not maintain follicle integrity, with the hope of improving oocyte development. We report that an alginate-based follicle culture system supports both follicular and oocyte growth in vitro, with little effect on the oocyte transcriptome. Nevertheless, oocytes obtained from these follicles exhibit an increased incidence of defects in spindle formation and chromosome alignment as well as pronounced abnormalities in cortical granule biogenesis. Developmental competence is also highly compromised, because few matured oocytes develop into 1-cell embryos with pronuclei. This situation contrasts with a high incidence of pronuclear formation following development using an existing in vitro culture system that does not preserve follicle integrity.