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Background: MicroRNAs (miRNAs) are presented in the uterine lumen of many mammals, and in vitro experiments have determined that several miRNAs are important for the regulation of endometrial and trophoblast functions. Our aim was to identify and contrast the miRNAs present in extracellular vesicles (EVs) in the uterine lumen fluid (ULF) at the onset of attachment in cattle pregnancies (gestation d 18) initiated by artificial insemination (AI) or by the transfer of an in vitro-produced blastocyst (IVP-ET). A third group had no conceptus after the transfer of an IVP embryo., Results: The abundance of 263 annotated miRNAs was quantified in the EVs collected from ULF. There was an increase in the transcript abundance of 20 miRNAs in the ULF EVs from the AI pregnant group, while 4 miRNAs had a lower abundance relative to the group not containing a conceptus. Additionally, 4 miRNAs were more abundant in ULF EVs in the AI pregnant group relative to IVP-ET group (bta-mir-17, bta-mir-7-3, MIR7-1, MIR18A). Specific miRNAs in the ULF EVs were co-expressed with messenger RNAs expressed in extra-embryonic tissues and endometrium, including genes that are known to be their targets., Conclusions: The results provide biological insights into the participation of miRNAs in the regulation of trophoblast proliferation and differentiation, as well as in endometrium receptivity. The knowledge that in vitro cultured embryos can contribute to the altered abundance of specific miRNAs in the uterine lumen can lead to the development of corrective approaches to reduce conceptus losses during the first month of pregnancy in cattle., (© 2024. The Author(s).)

Background: Subfertility is a major issue facing the dairy industry as the average US Holstein cow conception rate (CCR) is approximately 35%. The genetics underlying the physiological processes responsible for CCR, the proportion of cows able to conceive and maintain a pregnancy at each breeding, are not well characterized. The objectives of this study were to identify loci, positional candidate genes, and transcription factor binding sites (TFBS) associated with CCR and determine if there was a genetic correlation between CCR and milk production in primiparous Holstein cows. Cows were bred via artificial insemination (AI) at either observed estrus or timed AI and pregnancy status was determined at day 35 post-insemination. Additive, dominant, and recessive efficient mixed model association expedited (EMMAX) models were used in two genome-wide association analyses (GWAA). One GWAA focused on CCR at first service (CCR1) comparing cows that conceived and maintained pregnancy to day 35 after the first AI (n = 494) to those that were open after the first AI (n = 538). The second GWAA investigated loci associated with the number of times bred (TBRD) required for conception in cows that either conceived after the first AI (n = 494) or repeated services (n = 472)., Results: The CCR1 GWAA identified 123, 198, and 76 loci associated (P < 5 × 10 - 08 ) in additive, dominant, and recessive models, respectively. The TBRD GWAA identified 66, 95, and 33 loci associated (P < 5 × 10 - 08 ) in additive, dominant, and recessive models, respectively. Four of the top five loci were shared in CCR1 and TBRD for each GWAA model. Many of the associated loci harbored positional candidate genes and TFBS with putative functional relevance to fertility. Thirty-six of the loci were validated in previous GWAA studies across multiple breeds. None of the CCR1 or TBRD associated loci were associated with milk production, nor was their significance with phenotypic and genetic correlations to 305-day milk production., Conclusions: The identification and validation of loci, positional candidate genes, and TFBS associated with CCR1 and TBRD can be utilized to improve, and further characterize the processes involved in cattle fertility.

Background: Subfertility is one challenge facing the dairy industry as the average Holstein heifer conception rate (HCR), the proportion of heifers that conceive and maintain a pregnancy per breeding, is estimated at 55-60%. Of the loci associated with HCR, few have been validated in an independent cattle population, limiting their usefulness for selection or furthering our understanding of the mechanisms involved in successful pregnancy. Therefore, the objectives here were to identify loci associated with HCR: 1) to the first artificial insemination (AI) service (HCR1), 2) to repeated AI services required for a heifer to conceive (TBRD) and 3) to validate loci previously associated with fertility. Breeding and health records from 3359 Holstein heifers were obtained after heifers were bred by AI at observed estrus, with pregnancy determined at day 35 via palpation. Heifer DNA was genotyped using the Illumina BovineHD BeadChip, and genome-wide association analyses (GWAA) were performed with additive, dominant and recessive models using the Efficient Mixed Model Association eXpedited (EMMAX) method with a relationship matrix for two phenotypes. The HCR1 GWAA compared heifers that were pregnant after the first AI service (n = 497) to heifers that were open following the first AI service (n = 405), which included those that never conceived. The TBRD GWAA compared only those heifers which did conceive, across variable numbers of AI service (n = 712). Comparison of loci previously associated with fertility, HCR1 or TBRD were considered the same locus for validation when in linkage disequilibrium (D' > 0.7)., Results: The HCR1 GWAA identified 116, 187 and 28 loci associated (P < 5 × 10 - 8 ) in additive, dominant and recessive models, respectively. The TBRD GWAA identified 235, 362, and 69 QTL associated (P < 5 × 10 - 8 ) with additive, dominant and recessive models, respectively. Loci previously associated with fertility were in linkage disequilibrium with 22 loci shared with HCR1 and TBRD, 5 HCR1 and 19 TBRD loci., Conclusions: Loci associated with HCR1 and TBRD that have been identified and validated can be used to improve HCR through genomic selection, and to better understand possible mechanisms associated with subfertility.

The majority of pregnancy loss in ruminants occurs during the first three weeks after conception, particularly during the period of conceptus elongation that occurs prior to pregnancy recognition and implantation. This review integrates established and new information on the biological role of ovarian progesterone (P4), prostaglandins (PGs), interferon tau (IFNT) and cortisol in endometrial function and conceptus elongation. Progesterone is secreted by the ovarian corpus luteum (CL) and is the unequivocal hormone of pregnancy. Prostaglandins (PGs) and cortisol are produced by both the epithelial cells of the endometrium and the trophectoderm of the elongating conceptus. In contrast, IFNT is produced solely by the conceptus trophectoderm and is the maternal recognition of pregnancy signal that inhibits production of luteolytic pulses of PGF2α by the endometrium to maintain the CL and thus production of P4. Available results in sheep support the idea that the individual, interactive, and coordinated actions of P4, PGs, IFNT and cortisol regulate conceptus elongation and implantation by controlling expression of genes in the endometrium and/or trophectoderm. An increased knowledge of conceptus-endometrial interactions during early pregnancy in ruminants is necessary to understand and elucidate the causes of infertility and recurrent early pregnancy loss and provide new strategies to improve fertility and thus reproductive efficiency.

Background: We hypothesized that genes that are up-regulated in the uterine endometrium at the initiation of conceptus elongation in cattle, and that encode for secreted proteins, contribute to the composition of the uterine luminal fluid (ULF) and ultimately, drive conceptus elongation. The aims of this study were to: 1) screen endometrial transcriptomic data for genes that encode secreted proteins on Day 13; 2) determine temporal changes in the expression of these genes during the estrous cycle/early pregnancy; 3) determine if expression of these genes is affected by altered concentrations of progesterone (P4) in vivo and 4) determine if the protein products of these genes are detectable in ULF., Results: Of the fourteen candidate genes examined, quantitative real-time PCR analysis revealed the expression of APOA1, ARSA, DCN, LCAT, MUC13, NCDN, NMN, NPNT, NXPH3, PENK, PLIN2 and TINAGL1 was modulated in the endometrium (P<0.05) as the estrous cycle/early pregnancy progressed. APOA1, DCN and NPNT expression was higher in cyclic compared to pregnant heifers, and pregnancy increased (P<0.05) the expression of LCAT, NCDN, NMN, PLIN2 and TINAGL1. The magnitude of the increase in expression of APOA1, PENK and TINAGL1 on Day 13 was reduced (P<0.05) in heifers with low P4. Furthermore, low P4 decreased (P<0.05) the expression of LCAT and NPNT on Day 7, while an early increase (P<0.05) in the expression of NXPH3 and PLIN2 was observed in heifers with high P4. The protein products of 5 of the candidate genes (APOA1, ARSA, LCAT, NCDN and PLIN) were detected in the ULF on either Days 13, 16 or 19 of pregnancy., Conclusion: Using a candidate gene approach, we determined that both P4 concentration and the presence of the conceptus alter endometrial expression of PLIN2, TINAGL1, NPNT, LCAT, NMN and APOA1. Comparison of the expression profiles of these genes to proteins detected in ULF during conceptus elongation (i.e., Days 13 through 19) revealed the presence of APOA1, ARSA, LCAT, NCDN as well as members of the PLIN family of proteins that may play roles in driving conceptus elongation in cattle.

Establishment and maintenance of pregnancy in the pig involves intricate communication between the developing conceptuses and the maternal endometrium. This process occurs during trophoblast elongation which is spaciotemporally associated with conceptus synthesis and release of IL1B concomitant with pregnancy-specific endometrial up-regulation of IL-1 receptors, providing the potential for activation of the transcription factor, NFKB. The objective of the current investigation was to determine changes in expression and cellular localization of NFKB and associated factors during the estrous cycle and early pregnancy in the pig. In situ hybridization was used to localize changes in PGR, ESR1, and TNFRSF11A during the peri-implantation period. Quantitative RT-PCR was utilized to demonstrate gene expression changes for NFKB1, RELA, TNFRSF11A, TLR4, NFKBIA and NFKBIB. Transcription factor ELISA demonstrated an overall increase in RELA during the peri-implantation period in both cyclic and pregnant gilts. While the presence of TNFSF11A and TLR4 were both detected, TLR4 expression changes were temporally associated with NFKB expression and activation. Collectively, these data demonstrate that NFKB activation may occur during the period of uterine receptivity in both the cyclic and pregnant endometrium.

Establishment and maintenance of pregnancy results from signaling by the conceptus (embryo/fetus and associated extraembryonic membranes) and requires progesterone produced by the corpus luteum (CL). In most mammals, hormones produced by the trophoblast maintain progesterone production by acting directly or indirectly to maintain the CL. In domestic animals (ruminants and pigs), hormones from the trophoblast are antiluteolytic in that they act on the endometrium to prevent uterine release of luteolytic prostaglandin F2 alpha (PGF). In cyclic and pregnant sheep, progesterone negatively autoregulates expression of the progesterone receptor (PR) gene in the endometrial luminal (LE) and superficial glandular epithelium (GE). Available evidence in cyclic sheep indicates that loss of the PR is closely followed by increases in epithelial estrogen receptors (ER) and then oxytocin receptors (OTR), allowing oxytocin to induce uterine release of luteolytic PGF pulses. In pregnant sheep, the conceptus trophoblast produces interferon tau (IFN tau) that acts on the endometrium to inhibit transcription of the ER alpha gene directly and the OTR gene indirectly to abrogate development of the endometrial luteolytic mechanism. Subsequently, sequential, overlapping actions of progesterone, IFN tau, placental lactogen (PL) and growth hormone (GH) comprise a hormonal servomechanism that regulates endometrial gland morphogenesis and terminal differentiated function to maintain pregnancy in sheep. In pigs, the conceptus trophoblast produces estrogen that alters the direction of PGF secretion from an endocrine to exocrine direction, thereby sequestering luteolytic PGF within the uterine lumen. Conceptus estrogen also increases expression of fibroblast growth factor 7 (FGF-7) in the endometrial LE that, in turn, stimulates proliferation and differentiated functions of the trophectoderm, which expresses the FGF-7 receptor. Strategic manipulation of these physiological mechanisms can offer therapeutic schemes to improve uterine capacity, conceptus survival and reproductive health.

In sheep, the uterus produces luteolytic pulses of prostaglandin F2alpha (PGF) on Days 15 to 16 of estrous cycle to regress the corpus luteum (CL). These PGF pulses are produced by the endometrial lumenal epithelium (LE) and superficial ductal glandular epithelium (sGE) in response to binding of pituitary and/or luteal oxytocin to oxytocin receptors (OTR) and liberation of arachidonic acid, the precursor of PGF. Cyclooxygenase-one (COX-1) and COX-2 are rate-limiting enzymes in PGF synthesis, and COX-2 is the major form expressed in ovine endometrium. During pregnancy recognition, interferon tau (IFNtau), produced by the conceptus trophectoderm, acts in a paracrine manner to suppress development of the endometrial epithelial luteolytic mechanism by inhibiting transcription of estrogen receptor alpha (ERalpha) (directly) and OTR (indirectly) genes. Conflicting studies indicate that IFNtau increases, decreases or has no effect on COX-2 expression in bovine and ovine endometrial cells. In Study One, COX-2 mRNA and protein were detected solely in endometrial LE and sGE of both cyclic and pregnant ewes. During the estrous cycle, COX-2 expression increased from Days 10 to 12 and then decreased to Day 16. During early pregnancy, COX-2 expression increased from Days 10 to 12 and remained higher than in cyclic ewes. In Study Two, intrauterine infusion of recombinant ovine IFNtau in cyclic ewes from Days 11 to 16 post-estrus did not affect COX-2 expression in the endometrial epithelium. These results clearly indicate that IFNtau has no effect on expression of the COX-2 gene in the ovine endometrium. Therefore, antiluteolytic effects of IFNtau are to inhibit ERalpha and OTR gene transcription, thereby preventing endometrial production of luteolytic pulses of PGF. Indeed, expression of COX-2 in the endometrial epithelia as well as conceptus is likely to have a beneficial regulatory role in implantation and development of the conceptus.