Effects of Fertility on Gene Expression and Function of the Bovine Endometrium Megan A. Minten 1 , Todd R. Bilby 2 , Ralph G. S. Bruno 3 , Carolyn C. Allen 1 , Crystal A. Madsen 4 , Zeping Wang 1 , Jason E. Sawyer 5 , Ahmed Tibary 6 , Holly L. Neibergs 1 , Thomas W. Geary 4 , Stefan Bauersachs 7 , Thomas E. Spencer 1 * 1 Department of Animal Sciences, Washington State University, Pullman, Washington, United States of America, 2 Texas AgriLife Research and Extension, Stephenville, Texas, United States of America, 3 West Texas A&M University and Texas AgriLife Research and Extension, Amarillo, Texas, United States of America, 4 USDA-ARS, Fort- Keogh, Miles City, Montana, United States of America, 5 Department of Animal Science, Texas A&M University, College Station, Texas, United States of America, 6 Department of Veterinary Clinical Sciences, College of Veterinary Medicine and Center for Reproductive Biology, Washington State University, Pullman, Washington, United States of America, 7 Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-Universita ¨t Mu ¨ nchen, Munich, Germany Abstract Infertility and subfertility are important and pervasive reproductive problems in both domestic animals and humans. The majority of embryonic loss occurs during the first three weeks of pregnancy in cattle and women due, in part, to inadequate endometrial receptivity for support of embryo implantation. To identify heifers of contrasting fertility, serial rounds of artificial insemination (AI) were conducted in 201 synchronized crossbred beef heifers. The heifers were then fertility classified based on number of pregnancies detected on day 35 in four AI opportunities. Heifers, classified as having high fertility, subfertility or infertility, were selected for further study. The fertility-classified heifers were superovulated and flushed, and the recovered embryos were graded and then transferred to synchronized recipients. Quantity of embryos recovered per flush, embryo quality, and subsequent recipient pregnancy rates did not differ by fertility classification. Two in vivo-produced bovine embryos (stage 4 or 5, grade 1 or 2) were then transferred into each heifer on day 7 post-estrus. Pregnancy rates were greater in high fertility than lower fertility heifers when heifers were used as embryo recipients. The reproductive tracts of the classified heifers were obtained on day 14 of the estrous cycle. No obvious morphological differences in reproductive tract structures and histology of the uterus were observed in the heifers. Microarray analysis revealed differences in the endometrial transcriptome based on fertility classification. A genome-wide association study, based on SNP genotyping, detected 7 moderate associations with fertility across 6 different chromosomes. Collectively, these studies support the idea that innate differences in uterine function underlie fertility and early pregnancy loss in ruminants. Cattle with defined early pregnancy success or loss is useful to elucidate the complex biological and genetic mechanisms governing endometrial receptivity and uterine competency for pregnancy. Citation: Minten MA, Bilby TR, Bruno RGS, Allen CC, Madsen CA, et al. (2013) Effects of Fertility on Gene Expression and Function of the Bovine Endometrium. PLoS ONE 8(8): e69444. doi:10.1371/journal.pone.0069444 Editor: Xiaoqin Ye, The University of Georgia, United States of America Received April 22, 2013; Accepted June 8, 2013; Published August 5, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This project was supported in part by grant no. 1 R01 HD072898 from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: thomas.spencer@wsu.edu Introduction Infertility and subfertility are important and pervasive problems in both domestic animals and humans, and the greatest limitation to reproductive efficiency across mammalian species is embryonic mortality [1]. Embryo survival is a major factor affecting production and economic efficiency in all systems of meat and milk production by ruminants [2–4]. In beef cattle, estimates indicate that fertilization rate for oocytes is 90%, whereas average calving rates to a single service are between 40% and 55%, suggesting a rate of embryonic/fetal mortality (excluding fertiliza- tion failure) of about 35% to 50% [5]. The majority of embryonic loss (70–80%) occurs in the first 3 weeks of pregnancy [6], particularly between days 7 and 16 of pregnancy [7–9]. Further, embryo mortality is greater in non-lactating cows than heifers [7], and early pregnancy loss is even greater in high producing lactating dairy cattle and can approach 70% [2,10,11]. Infertility and subfertility are also major cost factors in the cattle embryo transfer industry [12]. Mean survival rate to calving following transfer of in vivo-derived embryos from superovulated donors is only 43% with a range from 31% to 60% [13], whereas the mean survival rate after transfer of in vitro-produced (IVP) embryos is lower and ranges from 30% to 40% [4,13]. Pregnancy loss is the most common complication of human gestation, occurring in as many as 75% of all women trying to have children. Roughly one- half of conceptions in humans result in pregnancy loss, with losses occurring most frequently in the first two weeks of gestation [14]. The failure to establish pregnancy in humans and animals is due to both embryonic and maternal factors [13,15,16]. Many of the pregnancy losses observed in natural or assisted reproductive technology pregnancies can be attributed to inadequate endome- trial receptivity, which can be defined as the physiological state of the uterus when conceptus growth and implantation for establish- ment of pregnancy is possible. However, knowledge of the PLOS ONE | www.plosone.org 1 August 2013 | Volume 8 | Issue 8 | e69444