Luteal Maintenance Research Articles

Research Progress on the Impact of Human Chorionic Gonadotropin on Reproductive Performance in Sows.

Human chorionic gonadotropin is a glycoprotein hormone produced by human or humanoid syncytiotrophoblasts that differentiate during pregnancy. Due to its superior stability and long-lasting effects compared to luteinizing hormone, it is often used to replace luteinizing hormone to regulate reproductive performance in sows. Human chorionic gonadotropin promotes oocyte maturation, follicle development, and luteinization, thereby increasing conception rates and supporting early embryonic development. In sow reproductive management, the application of human chorionic gonadotropin not only enhances ovulation synchrony but also improves the success rate of embryo implantation by regulating endometrial receptivity and immune mechanisms, significantly enhancing overall reproductive performance. This article primarily reviews the application of human chorionic gonadotropin in sow follicle development, luteal maintenance, and embryo implantation, providing theoretical support for its use in improving reproductive performance in sows.

Profiles of interferon-stimulated genes in multiple tissues and circulating pregnancy-associated glycoproteins and their association with pregnancy loss in dairy cows†.

Pregnancy loss (PL) in lactating dairy cows disrupts reproductive and productive efficiency. We evaluated the expression of interferon-stimulated genes (ISG) in blood leukocytes, vaginal and cervical epithelial cells, luteolysis-related genes, progesterone, and pregnancy-associated glycoprotein (PAG) profiles in lactating dairy cows (n = 86) to gain insight about PL. Expression of ISG on d17, d19, and d21 was greater in cows that maintained the pregnancy (P33) compared to nonpregnant with no PL (NP). Greater ISG differences between groups were observed in the cervix (96.7-fold) than vagina (31.0-fold), and least in blood leukocytes (5.6-fold). Based on individual profiles of ISG and PAG, PL was determined to occur either before (~13%) or after (~25%) d22. For cows with PL before d22, ISG expression was similar on d17 but by d21 was lower and OXTR was greater than P33 cows and similar to NP; timing of luteolysis was similar compared to NP cows suggesting embryonic failure to promote luteal maintenance and to attach to the endometrium (no increase in PAG). For cows with PL after d22, ISG expression was similar to P33 cows on d17, d19, and d21 and luteolysis, when it occurred, was later than NP cows; delayed increase in PAG suggested later or inadequate embryonic attachment. In conclusion, PL before d22 occurred due to embryonic demise/failure to signal for luteal maintenance, as reflected in reduced ISG expression by d21. Alternatively, embryos with PL between d22 and 33 adequately signaled for luteal maintenance (ISG) but had delayed/inadequate embryonic attachment and/or inappropriate luteolysis causing PL.

Early pregnancy associated changes in systemic lipid profiles of mares revealed by high resolution mass spectrometry

Early pregnancy in the mare presents a risk of embryo loss, with little known of the systemic lipid dynamics that contribute to a successful or failed pregnancy. Observations in horses and other species suggest that pregnancy is characterized by an anabolic (i.e. fat deposition) lipid profile initially, followed by a catabolic (lipid mobilization) profile toward the end of gestation. There have been no investigations of lipid metabolism in the very early stages of pregnancy, leading up to and during the time of maternal recognition. High resolution lipidomics presents an unprecedented opportunity to map the changes in lipid profiles of pregnant mares, to examine metabolic pathways contributing to establishment of pregnancy and to identify biomarkers for detection and monitoring of early pregnancy and pregnancy loss. Blood samples were collected from Thoroughbred mares in a commercial breeding operation at day 7 and day 14 following ovulation. Mares were live-covered, and pregnancy tested by transrectal ultrasound at day 14, hereby designated pregnant or non-pregnant. High resolution lipidomics analysis was performed on isolated plasma to compare profiles of pregnant (7P, 14P) and non-pregnant (7NP, 14NP) mares at day 7 and day 14 (total n = 72, split evenly in groups). Lipids were extracted using ice-cold butanol/methanol (1:1 v/v) with 10 mM ammonium acetate containing internal standards.LC separation was performed on a Phenomenex Kinetex C18 Column (2.6 µm, 100 Å, 100 × 2.1 mm) using 5 µL injection volume. The samples were analyzed in data dependent mode using Zeno EAD IDA on the ZenoTOF 7600 system. All data were analyzed using MS-DIAL 5.1. The analysis yielded a total of 10034 unique lipid species across all samples. A total of 277 and 1722 lipids were significantly altered (p<0.05) in pregnant vs non-pregnant animals at day 7 and day 14, respectively. Phosphatidylcholines (PC) were significantly increased in 7P vs 7NP, and phosphatidylethanolamines (PE) were significantly decreased in 7P vs 7NP, with similar trends observed at day 14. Lysophosphatidylcholines (LPC) were increased in 14P vs 14NP. PC and LPC concentrations also increased from day 7 to day 14, but only in pregnant animals. Collectively, these observations point to a major role for phospholipid metabolism in early pregnancy and are consistent with PE being a substrate for methylation to PC, which is a key source of choline crucial for early embryonic development. The roles of PC biosynthesis and supplementation in early equine pregnancy warrant further investigation. Furthermore, alterations in the lipid profile of mares at day 7 post-ovulation challenge the current paradigm that no systemic changes occur in the pregnant mare prior to classical maternal recognition and luteal maintenance.

Review: Maintenance of the ruminant corpus luteum during pregnancy: interferon-tau and beyond.

This manuscript reviews the mechanisms that maintain the corpus luteum (CL) of pregnancy in ruminants. In mammals, ovulation and luteinization of the remaining cells in the CL are due to a surge in Luteinizing Hormone (LH). In cattle, continued secretion of pulses of LH is essential for full development and function of the CL during the estrous cycle (LH pulses), however, the few studies on the CL after d20 of pregnancy do not indicate that LH is essential for maintaining the CL of pregnancy. The first essential step in maintaining the CL of pregnancy in ruminants is overcoming the mechanisms that cause regression of the CL in non-pregnant ruminants (d18-25 in cattle; d13-21 in sheep). These mechanisms have a uterine component involving oxytocin-induced prostaglandin F2α (PGF2A) pulses and a luteal component involving decreased progesterone production and luteal cell death. There is a critical role for embryonic interferon-tau (IFNT) in suppressing the uterine secretion of PGF2A during early pregnancy (d13-21 in sheep; d16-25 in cattle) and preventing luteolysis. There are also effects of IFNT on the expression of interferon-stimulated genes in other tissues including the CL but the physiologic role of these interferon-stimulated genes is not yet clear. After the IFNT period, there is another mechanism that maintains the CL of pregnancy in ruminants since embryonic IFNT is inhibited as attachment occurs and trophoblastic binucleate/giant cells begin secretion of pregnancy-associated glycoproteins. The second mechanism for luteal maintenance has not yet been defined but acts in a local manner (ipsilateral to pregnancy), and remains functional from d25 until just before parturition. The most likely mechanisms mediating later maintenance of the CL of pregnancy are increased uterine blood flow or decreased prostaglandin transporter expression in the utero-ovarian vasculature, preventing PGF2A reaching the CL. Finally, implications of these ideas on pregnancy loss in cattle are explored, highlighting the importance of inappropriate regression of the CL of pregnancy as a mechanism for pregnancy loss in cattle.

Pregnancy-induced changes in the transcriptome of the bovine corpus luteum during and after embryonic interferon-tau secretion†.

Understanding luteal maintenance during early pregnancy is of substantial biological and practical importance. Characterizing effects of early pregnancy, however, has historically been confounded by use of controls with potential exposure to early Prostaglandin F2-alpha (PGF) pulses or differences in Corpus Luteum (CL) age. To avoid this, the present study utilized bihourly blood sampling to ensure control CL (n=6) were of a similar age to CL from pregnant animals (n=5), yet without exposure to PGF pulses. Additionally, CL from second month of pregnancy (n=4) were analyzed to track fate of altered genes after cessation of embryonic interferon tau (IFNT) secretion. The major alteration in gene expression in first month of pregnancy occurred in interferon-stimulated genes (ISGs), with immune/interferon signaling pathways enriched in three independent over-representation analyses. Most ISGs decreased during second month of pregnancy, though, surprisingly, some ISGs remained elevated in the second month even after cessation of IFNT secretion. Investigation of luteolytic genes found few altered transcripts, in contrast to previous reports, likely due to removal of controls exposed to PGF pulses. An exception to this trend was decreased expression of transcription factor NR4A1. Beyond luteolytic genes and ISGs, over representation analyses highlighted the prevalence of altered genes within the extracellular matrix and regulation of Insulin-like growth factor (IGF) availability, confirming results of other studies independent of luteolytic genes. These results support the idea that CL maintenance in early pregnancy is related to lack of PGF exposure, although potential roles for CL expression of diverse ISGs and other pathways activated during early pregnancy remain undefined.

Antiluteolytic mechanisms and the establishment of pregnancy in the pig

Extended exposure of progesterone and conceptus estrogen influences the vascular compartment of the uterus and expression of many factors, such as prostaglandins (PGs), growth factors, extracellular matrix and adhesion molecules, cytokines and transcription factors. One of the supportive mechanisms by which the conceptus inhibits luteolysis is by changing PG synthesis in favor of luteoprotective PGE2. Alteration in PG synthesis may result from increased PGE synthase (mPGES-1) expression in the trophoblast and endometrium on days 10-13 of pregnancy with simultaneous down-regulation of PGF synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1). Conceptus and endometrial, rather than luteal, synthesis of PGE2, is involved in the process of maternal recognition of pregnancy. However, complex (direct and indirect) actions of estrogen on the CL, including decreased luteal VEGF soluble receptor on day 12 of pregnancy, are important for luteal maintenance. Moreover, conceptus signals affect another lipid signaling component - lysophosphatidic acid receptor (LPA3), as well as HoxA10 and Wnt in the endometrium, to create the appropriate uterine environment for establishment of pregnancy and implantation.

Effects of Equine Chorionic Gonadotropin on Ovulatory and Luteal Characteristics of Mares Submitted to an P4-Based Protocol of Ovulation Induction With hCG

The aim of this study was to evaluate the effect of equine chorionic gonadotropin (eCG) at the end of progesterone (P4) treatment on follicular and luteal characteristics during transition period (TP) and reproductive breeding season (RP). A total of 13 crossbred mares were distributed in two experimental groups in the spring and summer (n = 26). The animals received intravaginal P4 (1.9 g) releasing device from D0 to D10. On removal of P4 device, the mares received 400 IU of eCG (eCG group) or saline solution (control group). Human chorionic gonadotropin (hCG; 1.750 IU) was administered (DhCG) as soon as ovulatory follicle (OF) ≥35 mm was detected. Ovarian ultrasonography was performed from D0 until 15 days after ovulation. Blood samples were collected on D0, D5, D10, DhCG, 9 days after ovulation (CL9D), and 13 days after ovulation (CL13D). P4 and estradiol concentrations were assessed by chemiluminescence. Data were compared by Tukey test at P < .05. Ovulation rate was similar (P = .096) between seasons (RP = 100%; TP = 70%) but occurred earlier (P = .015) in RP (34.8 ± 10.1 hours) compared with TP (42.0 ± 10.4 hours). Interactions between season and treatment were observed for OF diameter (mm) (RP/control = 36.2 ± 1.8ab; RP/eCG = 32.9 ± 2.8 b; TP/control = 32.2 ± 1.2 b; TP/eCG = 37.2 ± 1.9a; P = .004) and for corpus luteum (CL) diameter (mm) on CL13D (RP/control = 25.4 ± 3.5a; RP/eCG = 22.5 ± 1.8ab; TP/control = 21.6 ± 4.9 b; TP/eCG = 27.4 ± 4.3a; P = .023), although no differences were observed for serum P4 on CL13D (RP/control = 6.0 ± 3.1 ng/mL; RP/eCG = 5.8 ± 0.9 ng/mL; TP/control = 3.6 ± 2.7 ng/mL; TP/eCG = 5.1 ± 2.3 ng/mL; P = .429) or for day of structural CL regression (RP/control = 12.8 ± 1.9; RP/eCG = 12.1 ± 1.1; TP/control = 11.0 ± 1.7; TP/eCG = 13.2 ± 2.0; P = .102). The application of eCG at the moment of P4 implant removal seemed to increase the capacity of luteal maintenance during spring TP. However, eCG treatment was worthless during the breeding season.

Functions of Lysosomes in Mammalian Female Reproductive System

The lysosome is the most acidic membrane-bound intracellular organelle. Lysosomal acidity is primarily maintained by vacuolar H+-ATPase (V-ATPase) and counter ion channels. There are >60 hydrolytic enzymes in the lysosome for its fundamental digestive role. Lysosomes also play important roles in endocytosis, exocytosis, autophagy, and cell death. Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here. In the ovary, lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis, regulation of follicular atresia, follicle rupture during ovulation, luteal cell survival, and luteal regression. In the oviduct, lysosomes are involved in endocytosis of both serum and oviductal luminal components. In the uterus during the menstrual/estrous cycle, lysosomes are associated with endometrial secretion, apoptosis, and menstruation. In the uterus during early pregnancy, lysosomes are involved in the temporal and directional changes of endocytosis, uterine epithelial acidification upon embryo implantation initiation, and embryo-maternal mutual communications via extracellular vesicles. In the placenta, lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition. In the mammary gland, lysosomes are important for mammary gland development and involution. These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction, from ovulation to ovarian steroidogenesis for pregnancy maintenance, and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications, to optional postpartum nurturing of newborns via lactation. Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.

Inhibition of luteolysis and embryo-uterine interactions during the peri-implantation period in pigs

Inhibition of luteolysis and establishment of pregnancy in pigs results from oestrogen secretion by the conceptuses and requires progesterone produced by the corpus luteum (CL). An integral part of maternal recognition of pregnancy in the pig is the redirection of prostaglandin (PG) F2alpha secretion from endocrine (blood) to exocrine (uterus) direction and an increase of PGE2 synthesis in both the endometrium and conceptus. Uterine and conceptus PGE2 synthases play an integrated role in establishing the PGE2:PGF2alpha ratio necessary for luteal maintenance. The luteolytic or luteotrophic changes in the CL are synchronised with the release of maternal pituitary and ovarian hormones. The presence of uterine oxytocin (OT) and luteinising hormone (LH) receptors are important for the luteolytic effect of PGF2alpha. Conceptus oestrogen secretion coincides with autocrine and paracrine dialogue between the multiple conceptuses and uterine biological compounds and their receptors in trophoblast and endometrium.

Using Estrous Behavior to Time Initiation of Oxytocin Administration to Prolong Luteal Function in Mares

The objective of this study was to use estrous behavior alone to determine the appropriate time for beginning an oxytocin treatment protocol for estrus suppression. We hypothesized that administration of oxytocin beginning 8 days after the onset of estrus will prolong the luteal phase in mares. Twenty-three light breed mares (aged 4–20 years) were exposed to a stallion and observed for signs of sexual receptivity. Mares not displaying signs received 250 μg of cloprostenol intramuscularly (IM) and were teased again 3–4 days later. On the day that estrous behavior was observed (Day 0), mares were randomly divided into two groups: oxytocin (n = 11): oxytocin (60 IU, IM) was administered once daily from Day 8–17; control (n = 12): did not receive treatment. Blood was collected from all mares every 4 days throughout Day 17, and every 7 days thereafter until Day 45. Serum progesterone concentrations >1.0 ng/mL were indicative of a functioning corpus luteum. Interestrus interval was defined as the period between Day 0 and the day when progesterone next reached <1.0 ng/mL. The average interestrus interval was higher for treated mares compared with control mares (32.4 ± 4.2 vs. 21.8 ± 1.5 days, respectively, P = .01). In the oxytocin group, the interestrus interval was longer than 31 days in 6 of 11 (54.5%) mares and up to 45 days in 5 of 11 mares (45.5%). We conclude that luteal maintenance beyond 30 days was attained by once-daily oxytocin administration beginning 8 days following behavioral estrus in a majority of mares.

Cyclooxygenase-2 is inhibited in prolonged luteal maintenance induced by intrauterine devices in mares

Treatment with intrauterine devices (IUD) prolongs luteal phases in mares, but the mechanism for this has not been fully elucidated. The aims of the present study were to examine how IUDs affect the uterus to induce longer luteal phases, particularly the role of cyclooxygenase-2 (COX-2) in the maintenance of the corpus luteum (CL). Twenty-seven reproductively normal mares were included: 12 were inseminated (AI), and 15 were fitted with IUDs. Blood samples for progesterone were obtained on Days 0, 3, 5, 7, 9, 11, 13, 14, and 15 (relative to day of ovulation). The groups were further divided into non-pregnant (AI-N, n = 4), pregnant (AI-P, n = 8), normal (IUD-N, n = 8) and prolonged luteal phase (IUD-P, n = 7) based on ultrasonic examinations and serum progesterone concentrations on Days 14 and 15. Blood sampling to quantify the PGF2α metabolite (PGFM) was performed through a catheter hourly from 15:00 to 20:00 h on Day 14, and from 6:00 until 13:00 h on Day 15. On Day 15, a low-volume uterine lavage followed by an endometrial biopsy was performed. Estradiol concentration in the Day 15 serum and lavage fluid was determined, while the abundance of COX-2 was evaluated in the biopsy specimens using western blotting (WB) and immunohistochemistry (IHC). All pregnant mares were negative for COX-2 in IHC samples and 5 of 8 were negative in WB samples while all mares of the IUD-N group were positive for COX-2. Of the seven mares in the IUD-P group, five and four were negative for COX-2 with the IHC and WB samples, respectively. The results from this study indicate that IUDs, when effective, suppress COX-2, leading to the inhibition of PGF2α release and maintenance of CL.

Endometrial prostaglandin synthases, ovarian steroids, and oxytocin receptors in mares with oxytocin-induced luteal maintenance

Oxytocin (OXT) has been used to prolong the luteal phase in mares, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of chronic exogenous OXT administration to mid-luteal phase mares on luteal maintenance. Also, endometrial expression of prostaglandin endoperoxide synthase 2 (PTGS2), prostaglandin F2α, E2 and I2 synthases (AKR1C3, PTGES, and PTGIS), oxytocin receptor (OXTR), progesterone receptor (PGR), and estrogen receptors 1 (ESR1) and 2 (ESR2) were assessed in mares experiencing luteal maintenance 2 weeks after chronic exogenous OXT administration. Control mares (n = 5; C group) received 6 mL of saline im, whereas OXT (60 units/mare) was administered im (n = 6; OXT group), every 12 hours, on days 7 to 14 postovulation. After endometrial biopsy in groups C (Day 10) and OXT (Day 24), luteolysis occurred within 3 or 6 days, respectively. Luteal maintenance took place in 4 of 6 (67%) of OXT-treated mares. Progesterone in C group was the highest on biopsy day (P < 0.05). In OXT mares, PTGS2, ESR1 (P < 0.05), PTGES, PTGIS, PGR, and ESR2 (P < 0.01) gene transcription decreased, whereas OXTR increased (P < 0.05) in comparison with the C group. In OXT-treated mares, endometrial ESR2 protein expression decreased (P < 0.05), but OXTR increased (P < 0.05) compared with control animals. In both experimental groups, PTGS2 was mainly immunolocalized in surface epithelium, whereas AKR1C3, PTGES, PTGIS, and PGR were in surface and glandular epithelia. ESR1 and ESR2 were found in glandular epithelium and OXTR in stromal cells. High immunolabeling for PTGES, PTGIS, PGR, and OXTR and low for ESR2 was detected in endometrium of OXT-group mares with extended diestrus. Prolonged luteal function associated with chronic OXT treatment may be related to different spatial expression of OXTR and PGR in the endometrium. The observed reduction of endometrial ESR2 may be responsible for the maintenance of PGR in luminal and glandular epithelium. Also, ESR2 may attenuate the transcriptional activity of ESR1 in mare endometrium. This study offers new knowledge on the endometrial expression of ovarian steroids and OXT receptors in OXT pharmacologically induced luteal maintenance in the mare.

Uterine expression of cytokines and contraction-associated genes in preterm birth of Brp-39 null mutant mice

Oxytocin (OXT) has been used to prolong the luteal phase in mares, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of chronic exogenous OXT administration to mid-luteal phase mares on luteal maintenance. Also, endometrial expression of prostaglandin endoperoxide synthase 2 (PTGS2), prostaglandin F2α, E2 and I2 synthases (AKR1C3, PTGES, and PTGIS), oxytocin receptor (OXTR), progesterone receptor (PGR), and estrogen receptors 1 (ESR1) and 2 (ESR2) were assessed in mares experiencing luteal maintenance 2 weeks after chronic exogenous OXT administration. Control mares (n = 5; C group) received 6 mL of saline im, whereas OXT (60 units/mare) was administered im (n = 6; OXT group), every 12 hours, on days 7 to 14 postovulation. After endometrial biopsy in groups C (Day 10) and OXT (Day 24), luteolysis occurred within 3 or 6 days, respectively. Luteal maintenance took place in 4 of 6 (67%) of OXT-treated mares. Progesterone in C group was the highest on biopsy day (P < 0.05). In OXT mares, PTGS2, ESR1 (P < 0.05), PTGES, PTGIS, PGR, and ESR2 (P < 0.01) gene transcription decreased, whereas OXTR increased (P < 0.05) in comparison with the C group. In OXT-treated mares, endometrial ESR2 protein expression decreased (P < 0.05), but OXTR increased (P < 0.05) compared with control animals. In both experimental groups, PTGS2 was mainly immunolocalized in surface epithelium, whereas AKR1C3, PTGES, PTGIS, and PGR were in surface and glandular epithelia. ESR1 and ESR2 were found in glandular epithelium and OXTR in stromal cells. High immunolabeling for PTGES, PTGIS, PGR, and OXTR and low for ESR2 was detected in endometrium of OXT-group mares with extended diestrus. Prolonged luteal function associated with chronic OXT treatment may be related to different spatial expression of OXTR and PGR in the endometrium. The observed reduction of endometrial ESR2 may be responsible for the maintenance of PGR in luminal and glandular epithelium. Also, ESR2 may attenuate the transcriptional activity of ESR1 in mare endometrium. This study offers new knowledge on the endometrial expression of ovarian steroids and OXT receptors in OXT pharmacologically induced luteal maintenance in the mare.

Novel concepts on the role of prostaglandins on luteal maintenance and maternal recognition and establishment of pregnancy in ruminants

In ruminants, the corpus luteum (CL) of early pregnancy is resistant to luteolysis. Prostaglandin (PG)E2 is considered a luteoprotective mediator. Early studies indicate that during maternal recognition of pregnancy (MRP) in ruminants, a factor(s) from the conceptus or gravid uterus reaches the ovary locally through the utero-ovarian plexus (UOP) and protects the CL from luteolysis. The local nature of the embryonic antiluteolytic or luteoprotective effect precludes any direct effect of a protein transported or acting between the gravid uterus and CL in ruminants. During MRP, interferon tau (IFNT) secreted by the trophoblast of the conceptus inhibits endometrial pulsatile release of PGF2α and increases endometrial PGE2. Our recent studies indicate that (1) luteal PG biosynthesis is selectively directed toward PGF2α at the time of luteolysis and toward PGE2 at the time of establishment of pregnancy (ESP); (2) the ability of the CL of early pregnancy to resist luteolysis is likely due to increased intraluteal biosynthesis and signaling of PGE2; and (3) endometrial PGE2 is transported from the uterus to the CL through the UOP vascular route during ESP in sheep. Intrauterine co-administration of IFNT and prostaglandin E2 synthase 1 (PGES-1) inhibitor reestablishes endometrial PGF2α pulses and regresses the CL. In contrast, intrauterine co-administration of IFNT and PGES-1 inhibitor along with intraovarian administration of PGE2 rescues the CL. Together, the accumulating information provides compelling evidence that PGE2 produced by the CL in response to endometrial PGE2 induced by pregnancy may counteract the luteolytic effect of PGF2α as an additional luteoprotective mechanism during MRP or ESP in ruminants. Targeting PGE2 biosynthesis and signaling selectively in the endometrium or CL may provide luteoprotective therapy to improve reproductive efficiency in ruminants.

Serum progesterone levels in early pregnant women and its relation to luteal maintenance therapy for pregnancy protection

Objective To investigate serum levels of β-human chorionic gonadotrophin (β-HCG) and progesterone (P) in early pregnant women, and their relation to luteal maintenance therapy for early pregnancy protection. Methods One hundred and thirty five infertility women treated in Department of Gynecology and Obstetrics of Beijing Chaoyang Hospital from January 2007 to December 2011. Total 150 pregnancy cycles, including 84 with intrauterine insemination (IUI) and 66 with natural conception, were divided into two groups: normal intrauterine pregnancy group (group A, n=80) and early pregnancy loss group (group B, n=70). Medical history, ultrasonic findings, serum female hormone, P and β-HCG levels at early pregnancy stage were analyzed. Results The age of group A and group B was (30.0±3.9) years and (30.7±4.9) years, respectively (P>0.05). The follicle-stimulating hormone/luteinizing hormone in group A and group B was 1.57±0.96 and 1.56±1.08; the estradiol levels in two groups were (152±66) pmol/L and (147±69) pmol/L, respectively (both P>0.05). There were no differences in dominant follicles and endometrial thickness between groups A and B (P>0.05). Ovulation promotion and luteal support treatments were adopted in both groups: 50%(40/80) of cycles in group A received ovulation promotion, 73%(58/80) of cycles received luteal support, while 44%(31/70) and 76%(53/70) received in groups B, respectively. The levels of serum progesterone in group A during 14-21 d, 22-27 d and ≥28 d after ovulation were higher than those in group B at each time points (P 0.05). Conclusion Dynamic monitoring of serum progesterone level in early pregnancy may be used as an auxiliary index for prediction of pregnancy outcome, but it may not be an indicator for luteal maintenance therapy. Key words: Pregnancy trimester, first; Intrinsic factor; Abortion, spontaneous; Progesterone; Corpus luteum maintenance

113 PRELIMINARY CHARACTERIZATION OF OXYTOCINASE IN EQUINE SERUM

Oxytocinase/insulin regulated aminopeptidase (IRAP) or leucyl-cystinyl aminopeptidase (LNPEP) is an enzyme that is involved in the regulation of hormones such as oxytocin, vasopressin, and angiotensin in both humans and sheep. Historically, very low levels of this aminopeptidase were reported in monthly samples obtained from cycling and pregnant mares using an enzymatic colourimetric method. The regulation of oxytocin in horses is of interest because of its central role in uterine clearance, luteal maintenance, parturition, passage of fetal membranes, maternal foal bonding, and milk let-down. A preliminary study was performed with the objective of re-examining the level of serum oxytocinase in nonpregnant control (n = 3 mares sampled every other day; EOD of the oestrous cycle), and n = 5 mares sampled Day 12 to 15; oxytocin-treated (n = 2 mares sampled Day 12 to 15), and early pregnant mares (n = 6 mares sampled EOD), using more sensitive ELISA methodology. Mares were examined daily in oestrus until ovulation (Day 0) and from Days 10 to 21, using transrectal ultrasonography of the reproductive tract. Palpable changes in uterine and cervical tone, ultrasound measurement of dominant follicles, oedema scores (0 to 4 with 4 being maximal oedema), and changes in luteal echotexture and size were recorded. Pregnant mares were bred using AI (&gt;200 million motile and normal sperm) from a proven stallion while in oestrus until ovulation, beginning when the dominant follicle was &gt;35 mm. Oxytocin-treated mares were administered 60 IU IM oxytocin SID from Day 7 to 14. Blood was collected to obtain serum. Changes in serum oxytocinase levels were measured using a commercially available ELISA kit for Horse LNPEP according to the manufacturer's instructions (MyBioSource, San Diego, CA, USA) and validated for use in our laboratory using serial dilutions of pooled serum with an intra-assay and inter-assay CV &lt;15%. The lowest standard of the assay was 31.2 ng mL–1. Preliminary results of this pilot study demonstrated in control cycles median serum oxytocinase levels below the lowest standard (2.0 to 30.1 ng mL–1). Oxytocin-treated mares had median serum oxytocinase levels from 39 to 61 ng mL–1 on Days 12 to 15. Early pregnant mares had detectable levels from Day 8 to 21 (medians ranging from 40 to 89 ng mL–1). We concluded that serum oxytocinase levels were below the lowest standard in diestrus, and were low but detectable in oxytocin-treated mares. The highest oxytocinase levels were measured during early pregnancy. Further studies of serum oxytocinase in a larger population of mares, along with studies of tissue mRNA levels of oxytocinase, are required to better understand the regulation of oxytocin in horses. Research was supported by the Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada) and the Equine Health Research Fund (University of Saskatchewan, SK, Canada).

Mouse CD11c+ dendritic cells are crucial in folliculogenesis, luteinization, and luteal maintenance

Mouse CD11c+ dendritic cells are crucial in folliculogenesis, luteinization, and luteal maintenance

Effect of pregnancy on endometrial expression of luteolytic pathway components in the mare

Endometrial oxytocin receptors (OXTR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are central components of the luteolytic pathway in cyclic mares, and their suppression is thought to be critical to luteal maintenance during early pregnancy. We examined the effect of pregnancy on endometrial expression of potential regulators of prostaglandin (PG) F2α secretion in mares. Expression of the nuclear progesterone receptor and oestrogen receptor ERα was high during oestrus, and depressed when progesterone was elevated; the opposite applied to the membrane progesterone receptor. PTGS2 was upregulated on Day 14 of dioestrus, but not pregnancy. Although OXTR mRNA expression was not elevated on Day 14 of dioestrus, protein abundance was; this increase in OXTR protein was absent on Day 14 of pregnancy. Intriguingly, gene and protein expression for PTGS2 and OXTR increased markedly between Days 14 and 21 of pregnancy suggesting that, although initial avoidance of luteolysis during pregnancy involves their suppression, this is a transient measure that delays rather than abolishes luteolytic pathway generation. The only oxytocin-PGF2α feedback loop component downregulated on both Days 14 and 21 of pregnancy was the PGF2α receptor we propose that downregulation of the PGF2α receptor uncouples the oxytocin-PGF2α feedback loop, thereby preventing generation of the large PGF2α pulses required for luteolysis.

Diestrus administration of oxytocin prolongs luteal maintenance and reduces plasma PGFM concentrations and endometrial COX-2 expression in mares

The objectives were to: (1) evaluate the efficacy of varying intervals of oxytocin administration in preventing luteolysis in mares; (2) examine PGF2α release in mares experiencing prolonged diestrus secondary to oxytocin treatment; and (3) evaluate the endometrial expression of oxytocin receptor, estrogen receptor α, and prostaglandin synthesis enzymes after oxytocin administration. In experiment I, mares received oxytocin (60 IU, im) daily on Days 8 to 10, 8 to 12, or 8 to 14 postovulation, and control mares received sterile saline. Prolongation of diestrus was defined by elevation of serum progesterone >1.0 ng/mL through Day 30 postovulation. The proportion of mares experiencing prolonged cycles increased (P < 0.01) as the number of days of oxytocin administration increased. Oxytocin administration on Days 8 to 10, 8 to 12, and 8 to 14 prolonged luteal maintenance in 3/7, 4/7, and 6/7 mares respectively, compared with 0/7 control mares. Treated mares with prolonged diestrus had lower (P < 0.05) plasma PGFM concentrations at Day 16 than did mares with normal diestrus periods. In experiment II, endometrial biopsies from mares treated with oxytocin from Days 8 to 14 postovulation (N = 6) had reduced cyclooxygenase-2 expression (P < 0.05) compared with control mares (N = 6) as determined by quantitative reverse transcription polymerase chain reaction and immunohistochemical staining. Oxytocin administration prolonged luteal maintenance in mares, with an increasing number of mares responding to treatment as the number of days of oxytocin administration was increased beyond Day 8 postovulation. Luteal maintenance in mares was also associated with decreased plasma PGFM concentrations and reduced endometrial cyclooxygenase-2 expression.

Endocrine and Molecular Control of Luteal and Placental Function in Dogs: A Review

In the domestic dog (Canis familiaris), the corpus luteum (CL) is the only source of progesterone (P4) in non-pregnant and pregnant animals. The progesterone secretion profiles are almost identical in both conditions until the last third of the luteal phase when the gradual P4 decline turns into a steep drop in pregnant bitches, indicating the onset of parturition. Consequently, the length of the CL-phase in non-pregnant dogs exceeds the luteal lifespan in pregnant animals. The canine CL-function is regulated by many species-specific regulatory mechanisms, the most intriguing of which is the reported independence of gonadotropic support during the first third of dioestrus. Recently, PGE2 has been proposed as one of the most important luteotropic factors acting locally during this time, but afterwards prolactin (PRL) appears to be the main luteotropic factor. Luteal regression/luteolysis occurs, however, in spite of an increased gonadotropic support. Lately, by demonstrating the expression of PRL-receptor (PRLr), a new insight into possible regulatory mechanisms has indicated that the supply of P4 could be controlled upstream of the steroidogenic machinery at the level of PRLr expression and/or function, subsequently leading to the functional suppression of the steroidogenic machinery. An endogenous source of a luteolytic agent is apparently lacking, implicating the luteal regression in non-pregnant bitches as a passive, degenerative process even if the PGF2α-receptor is constitutively expressed in canine CL. This is in contrast to pregnant dogs in which prepartum luteolysis seems to be an active process of CL destruction by PGF2α of utero/placental origin targeting the luteal PGF2α-receptor.