Association of a missense mutation in the luteinizing hormone/choriogonadotropin receptor gene (LHCGR) with superovulation traits in Chinese Holstein heifers

Background： Upon binding luteinizing hormone in the ovary, the luteinizing hormone/choriogonadotropin receptor （LHCGR） is necessary for follicular maturation and ovulation, as well as luteal function. We detected mutations in the LHCGR gene and evaluated their association with superovulation. Methods： Using polymerase chain reaction-single strand sequencing, we examined polymorphisms in LHCGR and Chinese Holstein heifers. conformation polymorphism （PCR-SSCP） and DNA the genotypes associated with superovulation traits in 127 Results： A G/T polymorphism （ss52050737） in exon 11 was significantly associated with the total number of ova and the number of transferable embryos. Conclusions： LHCGR may be a new predictor for superovulation in Chinese Holstein heifers.

The Effect of GnRHa Induced Superovulation on Endometrial Morphology and Estrogen Receptor and Progesterone Receptor in Mouse

To evaluate the effect of GnRHa induced superovulation protocol on endometrial morphology and function. Material & Methods Forty ICR mice were randomly allocated into 4 groups, among them, 2 experimental groups were injected with GnRHa+HMG+hCG, another 2 groups were given saline of same volume as control group. The uterine tissues were investigated at 24 h and 48 h after administration (experimental group) or ovulation (control group).The endometrial thickness, the size of gland and glandular lumen, the total area of glandular cells, the average height of glandular epithelium were measured from routine histological slides using computerized image analysis. The SP immunohistochemistry techniques with monoclonal antibodies were employed to semi quantitatively analize the estrogen receptor (ER) and progesterone receptor (PR) in glandular cells. Results The endometrial thickness was not significantly different between experimental groups and control groups at 24 h and 48 h (P>0.05).The average area, perimeter, maximal diameter of single gland and glandular lumen, the total area, average height of glandular epithelium in experimental groups were significantly smaller than those of in control groups at equivalent time stages (all P<0.01). The asynchronous development of gland epithelium and stroma cells, namely, pesudostratified glandular epithelium and predecidual changes of stroma cells were seen at same time in experimental groups. The positive percentage (%) and expression intense of ER and PR in glandular epithelium cells were significantly lower in experimental groups than in control groups (P<0.05). Conclusion The protocol with GnRHa had a negative effect on endometrial histological structure and down regulated the express of ER and PR, suggesting that this protocol effect on the endometrial morphology and function and could not facilitate the formation of a physiologic endometrium completely, which may be one of the causes of low pregnancy rates.

Factors Influencing the Superovulation and Artificial Insemination of Sheep

[Objective] This study aimed to find out a method for low-cost and highly efficient sheep superovulation treatment and artificial insemination. [Method] The factors those probably influencing the results of conventional superovulation and insemination, such as combination of FSH hormone and sponge suppository, estrus interval, number of insemination, and ram individuals were analyzed. [Result] The combination of sponge suppository and FSH produced in Beijing exhibited the poorest effect to superovulation, significantly worse than that of other combinations （P0.01）. The FSH produced in Ningbo, combined with sponge suppository or CIDR produced better effect to superovulation. The superovulation effect was better when the interval from the last FSH injection to estrus was 12 h, significantly better than that when the interval was 36 h （P0.01）; and there was no difference in the superovulation results when the interval was 0, 12 and 24 h. The pregnancy rate of two artificial inseminations was significantly higher than that of only one insemination （P0.01）. Rams themselves had significant influence on fertilization results. [Conclusion] The combination of domestic FSH and domestic sponge suppository cost much less and dose not reduce the superovulation results. Better fertilization result can be obtained if the ewes are inseminated twice with the sperm those gave high pregnancy rate.

Relationship between heart girth, serum progesterone and superovulation response ofdonor Holstein cows

Objective: To determine effects of variations of heart girth, volume trait on embryo quality grade, and to explore the relationship between heart girth and circulating progesterone and correlations of circulating progesterone with embryo quality grade in superstimulated donor Holstein cows. Methods: Nineteen cows were subjected to a standard superstimulation protocol using follicle stimulating hormone. Blood samples were collected before superovulation, at insemination and at collection of embryos for progesterone analysis. Embryo quality grades were compared between high and low heart girth donors. Moreover, Pearson's correlations were determined between heart girth, progesterone and embryo quality. Results: Variation of heart girth was not associated with significant differences in embryo quality grade between high and low heart girth donor cows (P>0.05). However, we observed a significantly higher percentage of the third-grade embryo (24.09%) in low heart girth donors compared to 9.64% in high heart girth donors. Moreover, the percentage of the transferable embryo was numerically higher and that of the degenerated embryo was numerically lower in low heart girth cows. Donor cows with low response to superovulation (total structures ≤ 3) expressed numerically higher mean heart girth, compared to donors with high (total structures>15) and medium (total structures = 4-15) superovulation. Heart girth and body weight of donor cows were moderately correlated (r=0.45, P<0.05), but none was correlated with circulating progesterone at different sampling times except for a moderate correlation between body weight and progesterone at embryo collection (r=0.54, P=0.02). Circulating progesterone before superovulation was moderately correlated to the second-grade embryo (r=0.46, P<0.05) and to the third grade (r=0.52, P<0.05) embryo. Conclusions: Volume traits heart girth may influence the response of Holstein cows to superovulation;however, future studies with a higher number of cows are warranted to clarify significant influences.

Effects of anestrus dog serum on superovulation in rats and mice

Objective:To study the effects of anestrus dog serum in rodents (contains high amount of FSH) compared with two common superovulatory programs.Methods: Rats (n=30) at diestrus phase were evenly divided into pregnant mare serum gonadotrophins (PMSG) group (administrated with 30 IU PMSG, and 48 h later following by 25 IU hCG), recombinant follicle stimulating hormone (rFSH) group (reducing dose every 12 h from 5 to 1 IU, then following by 25 IU hCG) and anestrus dog serum group (reducing dose every 12 h from 0.6 to 0.1 mL, then following by 25 IU of hCG). Mice (n=30) were also evenly divided into PMSG group (5 IU PMSG used at 13 pm, and 48 h later following by 5 IU of hCG), rFSH group (from 13 pm, reducing dose every 12 h from 2.5 to 0.5 IU, then following by 5 IU hCG) and anestrous dog serum group (from 13 pm, reducing dose every 12 h from 0.100 to 0.025 mL, then following by 5 IU hCG). Mice and rats placed with males for 24 h after last injection. Histology samples of ovaries were prepared and the numbers of corpus lutea were counted on day 14 after mating. Results: In mice, the differences among mean number of corpus lutea in all groups (P=0.01) and between FSH and dog serum group were significant (P=0.0007). But no significant differences were found between the mean number of corpus lutea in dog serum and PMSG, or between FSH and PMSG groups. In rats, mean number of corpus lutea were significant differences among three groups (P=0.01), and between PMSG and dog serum groups (P=0.02). Conclusions: Superovulatory response in anestrus dog serum group is similar to PMSG group in mice, which is relatively similar to FSH in rats.

Superovulation of the Cloned Cattle Derived from Somatic Cells and the Transfer of the Vitrified-Thawed Embryos of the Cloning Cattle

In this experiment, it was designed to carry out superovulation on the two cloned cattles, vitrification and transfer of the embryos recovered from them. First of all, it was carried out vitrification on embryos obtained by IVF. Results showed that there were no significant differences between the blastocysts (obtained by IVF) vitrified in EPS10 and these in EPS20 on the resuscitative rate and the developmental rate. The hatched rate of the blastocysts vitrified in EPS10 (31.3%, 35/112) was significantly higher than that in EPS20 (12.2%, 13/107)(P<0.01), so EPS20 was selected as the vitrification solution to freeze the embryos recovered from the cloned cattle. After superovulation, six (four usable embryos) and ten (nine usable embryos) embryos were respectively recovered from Kangkang and Shuanghuang. Two embryos were selected from the recovered embryos of each cloned cattle to freeze in EPS20, subsequently thawed and transferred into luteal ipsilateral uterine horns of 4 Holstein recipient cows after synchronization of estrus, respectively. At last, one recipient cow (No. 9908) became pregnant and delivered one healthy calf (descendant of the cloned cattle-Shuangshuang). The results of this experi- ment show that the cloned cattle as well as common cattle had better response to the exotic FSH and better ability to multiovulation, the embryos recovered from the cloned cattle can be vitrificated.

Research on the Optimal Dose of FSH for Superovulation of Mongolian Cattle

In this study, purebred Mongolian cattle were treated with a series of technical means including superovulation, artificial insemination, non-surgical embryo flushing, embryo grading and embryo cryopreservation, aiming at exploring a set of mature, stable production and breeding program for purebred Mongolian cattle embryos. The results showed that the optimal dose of FSH for superovulation of bred Mongolian cattle was 410 IU; the optimal dose of FSH for superovulation of muhiparous Mongolian cattle was 440 IU. Bred Mongolian cattle exhibited higher average number of available embryos and embryo utilization rate compared with muhiparous Mongolian cattle, but the latter exhibited higher average number of embryos recovered.

Effects of controlled superovulation on the expression of HOXA10,integrin αvβ3 and LIF in mice during peri-implantation

Objective:To observe the expression of endometrial homologous box gene A10(HOXA10),Integrinαvβ3 and leukemia suppressor factor(LIF)in peri-implantation mice,and to investigate the effect of controlled superovulation(COH)on endometrial receptivity in mice.Methods:After COH model preparation and blastocyst transplantation,kunming pure-bred mature mice were randomly divided into natural blastocyst+natural pseudocyst group,COH blastocyst+natural pseudocyst group,natural blastocyst+COH pseudocyst group,and COH blastocyst+COH pseudocyst group.The pregnancy rate and average number of beds in each group were observed on day 6 of conception.Western blot and RT-PCR were used to detect the expression of HOXA10,Integrinααvβ3,LIF protein and mRNA in the endometria of mice on day 5 of conception.Results:The pregnancy rate and average number of beds in the COH group were lower than those in the natural receptor group.The expression of HOXA10,Integrinααvβ3 and LIF in endometrium was significantly lower than that of the natural receptor mice(P<0.01).Conclusion:Superovulation drugs down-regulate the expression of HOXA10,Integrinαvβ3 and LIF in endometrium during the implantation window,reduce endometrial receptivity,and lead to low clinical pregnancy rate.

Increased Oocyte Production after Acupuncture Treatment during Superovulation Process in Mice

Objective To investigate whether acupuncture treatment during superovulation process improves ovarian response and increases egg production. Methods ICR female mice aged 12 -5 weeks were divided into control group, anaesthesia group and acupuncture group. Female mice were injected intraperitoneally with pregnant mare＇s serum gonadotropin （PMSG）, followed by human chorionic gonadotropin （hCG） injection 56 h later. Anaesthesia group and acupuncture group were anaesthetized three times by injecting 10% nembutal solution according to 7.5-10.0μl/g weight. Acupuncture group was treated three times with puncture Sanyinjiao Points（SP6） under anaesthesia. After 17 h of hCG injection, eggs were recovered and ovaries were isolated. Matured eggs were counted, ovarian histology and expression of follicle stimulating hormone receptor（FSH-R） in ovary were analyzed. Results Acupuncture treatment statistically increased the number of ovulated eggs. Histological section showed that some matured follicles left in the ovaries of control and anaesthesia groups after ovulation. On the contrary, all matured follicles ruptured and converted into corpus lutea in Acupuncture group. Expression of FSH-R in ovary was decreased in acupuncture group compared with those of the two others. Conclusion Acupuncture treatment during superovulation process improves ovarian response so as to increase egg production. The positive effect of acupuncture may be associated with regulated FSH-R expression.

Anti-Müllerian Hormone and Its Utility in Cattle Reproduction

Reproductive biotechnologies offer us greater possibilities to improve animal genetics. However, the success of these depends on different factors such as the proper selection of the donor female. For this reason, endocrine markers have been used to evaluate the ovarian reserve, which allow a successful selection of donors. Recent research has shown, among other things, that concentrations greater than 0.130 ng/mL of anti-Mullerian hormone (also known as Muller-inhibiting substance, which is a member of the transforming growth factor beta superfamily of growth and differentiation factors) are related to donors of more than fifteen transferable embryos. Therefore, this review describes studies showing that the measurement of anti-Müllerian hormone concentrations, before superovulation programs, reduces the costs per embryo produced.

The Effect of n-Hexane on the Gonad Toxicity of Female Mice

Objective To investigate the toxic effects of n-hexane on the Ganod of female mice.Methods n-Hexane was administered to four groups of mice by inhalation at doses of 0,3.0,15.1,and 75.8 mL/m3 respectivelyfor five weeks.Each group consisted of 10 mice,of which half were injected in first with 10 IU of pregnant mare serum gonadotrophin（PMSG） on the 33rd days,and then with 10 IU of human chorionic gonadotrophin（HCG） 48 hrs later.After the treatment,mouse sera were sampled and ovulating hormone（LH）,follicle-stimulating hormone（FSH）,estradiol（E2）,and progesterone（P4） levels were measured by electrochemiluminescence immunoassays（ECLIA）.In each group,the right ovaries of the non-super-ovulated mice were stained with hematoxylin and eosin while ovaries on the left side were prepared with the TUNEL method in order to detect apoptotic cells.Results The duration of the diestrus stage decreased significantly（P0.05） in the 75.8 mL/m3 group.All super-ovulated mice in each treatment group produced fewer eggs than those in the control group（P0.05）.The number of follicles in ovaries in the 75.8 mL/m3 group was smaller compared with the control group（P0.05）.The serum P4 levels in each treatment group were lower than those in the control group（F=6.196,P0.01）.The cell apoptotic rate in the 75.8 mL/m3 group was higher（P0.05）.Conclusion n-Hexane may have directly mediated via alterations hormone secretion and promoted granulosal cell apoptotic,which may be one of the important mechanisms for n-hexane induced mouse ovary impairment.

Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4

Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons（breeding and non-breeding season） on superovulation and the imported exogenous gene on growth.Results： In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep.Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons（P 〉 0.05）. The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group（P 〈 0.05）. The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.Conclusions： Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.

Anti-Mullerian Hormone and Follicle Counts as Predictors of Superovulatory Response and Embryo Production in Beef Cattle

This study evaluated Anti-Mullerian hormone (AMH) and/or follicle counts as predictors of subsequent superovulatory response and embryo production in 79 beef cows. Before superovulation, 3 to 5 mm follicles presented on the ovaries of donor cows were counted, and blood was collected for measure of serum AMH. Across cows, serum AMH ranged from 0.013 to 0.898 ng/mL, with a mean of 0.293 ng/mL. The distribution of both AMH concentrations and follicle counts were divided into quartiles (AMH Q1 through Q4, with Q1 the lowest and Q4 the highest) for analysis. Donor cows in AMH Q4 had a greater (P < 0.001) number of 3 to 5 mm follicles at the start of superovulation than did donors in either Q1 or Q2. At embryo collection, cows in AMH Q3 and 4 had more (P < 0.001) palpable corpora lutea (CL) than cows in AMH Q1. The mean number of embryos recovered from donor cows in AMH Q4 was greater (P < 0.001) than those recovered from cows in either AMH Q1 or 2, but similar to that of AMH Q3. Analysis indicated that AMH was positively correlated (P < 0.001) with mean follicles (r = 0.458), CL (r = 0.452) and embryos recovered (r = 0.430). Donor cows with higher follicle counts (F Q3 and 4) at the start of superovulation had more (P < 0.001) palpable CL at embryo collection than donor cows in F Q1 or 2. More (P < 0.001) embryos were recovered from cows with the highest follicle counts (F Q4) as compared with cows having lower (F Q1 and 2) follicle counts. Results confirm that relative AMH concentration was positively correlated with number of small antral follicles in the ovaries of cows;both AMH measure and antral follicle counts might be used to predict subsequent superovulatory response in beef cows.

Comparison of Three Doses of Gonadotrophin-releasing Hormone Agonist (GnRH-a) in IVF-ET Treatment Cycles

Objective To investigate the optimal dose of GnRH-a, which is sufficient for down regulation of pituitary without over-suppression of ovarian function in IVF superovulation protocols Methods Sixty patients undertook IVF-ET because of tube factor and /or endometriosis. Patients were divided into three groups randomly. Group A: 25 patients received a single dose of 1.88 mg Decapeptyl; Group B: 20 patients had a single dose of 2.50 mg Decapeptyl; Group C: 15 patients had a single dose of 3.75 mg Decapeptyl. Results The LH levels were 2.00±0.77 IU/L in Group A, 1.05±0.44 IU/L and 0.95 ±0.48 IU/L in Group B and C respectively on the day of starting gonadotropin injection; 1.97±0.76 IU/L in Group A, 0.96±0.42 IU/L and 0.77±0.49 IU/L in Group B and C respectively on the day of hCG administration. The LH levels in Group B and Group C were significantly lower than those in Group A (P<0.01). The mean number of gonadotropin ampoules was higher in Group B (38.05±6.08 ampoules) and Group C (40.0±10.62 ampoules) than in Group A (29.96±4.98 ampoules) (P< 0.01). The duration of stimulation significantly increased in Group B (12.9±1.81 d) and Group C (13.6±2.63 d) as compared with that in Group A (11.24±1.36 d) (P<0.01). There were no differences among three groups in E_2 levels on the day of hCG administration, the number of retrieved oocytes, fertilization rate, cleavage rate or pregnancy rate. There was no presence of LH peak in all groups. Conclusion The use of 1.88 mg Decapeptyl provides successful rate similar to 2.50 mg and 3.75 mg Decapeptyl, resulting in the reduced amount of gonadotropin, the duration of stimulation and the cost of treatment. The dose of 1.88 mg Decapeptyl is sufficient for pituitary down-regulation of IVF program for Chinese women.

Harvesting of Mouse Embryos at 0.5 Dpc as a Tool to Reduce Animal Use: Data from C57BL/6J, B6*129 and FVB/NJ Strains

Superovulation is used to stimulate the production and release of large amounts of oocytes in mice by using two hormones that mimic FSH (PMSG) and LH (hCG) effects. Since superovulation can have a negative impact on oocyte and embryo development, this investigation aimed to compare two alternatives for 2-cells embryo collection in order to reduce the number of females and to benefit from the superovulation process. Data from mouse embryo collection from our facility was analyzed to compare the number of 2-cells embryos collected at 1.5 dpc and the number of 2-cells embryos obtained after overnight incubation of 1-cell embryos, collected at 0.5 dpc. Genetically modified mouse strains with a similar background (C57BL/6J, B6*129 and FVB/NJ) were analyzed and for strains at a C57BL/6J and B6*129 background, the number of 2-cells embryos obtained after incubation was significantly higher when compared to the number of 2-cells embryos collected at 1.5 dpc (1.4-fold and 1.7-fold, respectively). C57BL/6J wild type mice had similar results with a higher number of 2-cells embryos when collection was performed at 0.5 dpc followed by incubation (1.4-fold). These results can help the planning of 2-cells embryo harvesting by reducing the number of females needed for this procedure.

First Trimester Pregnancy Loss May Temporary Reduce the Ovarian Response during the Subsequent IVF Cycle

The aim of this study was to compare the in vitro fertilization (IVF) cycles ended by miscarriage with subsequent IVF cycles in relation to various IVF cycle parameters and pregnancy termination modalities. Comparison of pre-miscarriage to post-miscarriage IVF cycles parameters demonstrated that lower peak E2 levels (1087 ± 593 versus 1237 ± 676 pg/ml, respectively;p p p p p p < 0.05), and an increase in the conception rate (34.7% versus 42.2%, respectively) at the second post-miscarriage IVF cycle.

Using Anti-Müllerian Hormone (AMH) as a Predictor of Ova Production for Bovine Embryo Transfer

The objective of this study was to determine the relationship of anti-Müllerian hormone (AMH) to ova production in various breeds of cattle in an embryo transfer program. Various factors that were evaluated included breed type, age, weight, body condition and flush history as to their influence on total ova recovery. The superovulation regimen called for CIDR (Controlled Internal Drug Release) insertion and a 2 cc injection of Combo (25 mg progesterone and 1.25 mg of estradiol per mL (2 cc IM injection)) on day 0. Day 4, FSH treatments were initiated in both AM and PM with decreasing amounts over the next four days. Day 6, along with FSH, prostaglandin was given in the AM and PM. On Day 7, a final FSH injection and CIDR removal were done in the AM. This resulted in estrus and AI on day 8 and collection of ova on day 15. Results were drawn from analyzing 369 animals;Angus n = 25, Black Brangus n = 43, Red Brangus n = 53, Brahman n = 103, Beefmaster n = 112, and Wagyu n = 33. Age of donors ranged from 1.6 years to 15.4 years at collection with an average age of 7.16 years of age. Analysis of results demonstrated that age clearly had a significant (P 0.05) on ova production. More importantly, we observed the total ova production was significantly (P < 0.05) associated with the AMH concentration (the greater the AMH concentration, the greater number of ova per flush).

High Levels Production of Recombinant Human Activin A—Effect upon <i>in Vivo</i>Follicle Stimulation

Activin plays an important role in numerous physiological processes such as cell differentiation and remodeling, regeneration and repair of tissues from various organs, angiogenesis, morphogenesis of glandular organs, pluripotency and differentiation of stem cells, cell adhesion and apoptosis. It participates in reproductive processes like embryogenesis, in the expression of Follicle Stimulating Hormone and Luteinizing Hormone and maturation of ovarian follicles and therefore has application in the area of reproduction of vertebrates. Given the economic importance of activin, we develop an efficient and economical method for the production of recombinant human activin A (rACT), using as expression system the yeast Pichia pastoris. rACT showed biological activity as it induced, on submicromolar dose, the increase of ovarian mass and the ovulation process in a mammal model.

Alpase of Follicles during the Period before and after Ovulation in Superovulated Rabbits

The Alpas of follicles was studied systematically during the period before and after ovulation in superovulated rabbits. The locations and changes of follicular Alpase varied with the four kinds of physiological status before and after ovulation. Alpasc activity was the highest at oestrus and started to drop at the beginning of ovulation, then it increased 15 h later and decreased slightly 39 h afterwards the beginning of ovulation. Alpase was found on the follicular basement membrane at oestrus. The synthesis of fiber in fibcrblasts was connected with Alpasc activity.

针刺对超排卵大鼠RT-BM1及母胎免疫耐受的影响

Objective:This study aimed to evaluate the effects of acupuncture on embryo implantation and maternal–fetal immune tolerance in rats with ovarian hyperstimulation,identify the mechanisms of acupuncture treatment,and promote application of acupuncture in the field of assisted reproduction.Methods:Eighty female Wistar rats were randomized into normal(N),model(M),acupuncture treatment(A),and progesterone treatment(P)groups,with 20 rats in each group.An ovarian hyperstimulation model was established in groups M,A,and P by the peritoneal injection of pregnant mare serum gonadotropin(PMSG)combined with human chorionic gonadotropin(HCG).Bilateral“Sanyinjiao(SP6)”,“Zusanli(ST36)”and“Taichong(LR3)”as acupoints were needled in rats in group A.Rats in group P received an intramuscular injection of progesterone(4 mg/kg).All pregnant rats were treated for seven days.On the 8th day,the pregnant rats were sacrificed for testing.The expression levels of estrogen(E2)and progesterone(P4)in serum and their receptors in the endometrium,Th1 and Th2 cytokines in serum and endometrium,RT-BM1(the counterpart of HLA-G in rats)mRNA,Ly49 activating receptors(Ly49s3,Ly49s5)and inhibitory receptors(Ly49i3,Ly49i4,Ly49i5)on immunocytes in the maternal–fetal interface were detected using ELISA,Western blot,and real-time polymerase chain reaction(PCR).Results:Compared with group N,the pregnancy rate and the number of implanted embryos were significantly decreased in group M(P<0.05);E2 and P4 levels in serum,and the protein levels of ER-αand Progesterone Receptor(PGR)in the implantation site of endometrial tissue were significantly reduced in group M(P<0.05);the expression of INF-γand IL-2 in serum and endometrium were significantly increased,while the expression of IL-4,IL-6,and IL-10 in serum and endometrium were significantly decreased in group M(P<0.05).The mRNA levels of RT-BM1,Ly49i3,and Ly49i4 receptors in group M were significantly higher than those in group N,whereas the mRNA levels of Ly49i5 and Ly49s5 receptors in group M were significantly lower than those in group N at the maternal–fetal interface(P<0.05).The pregnancy rate and number of implanted embryos were significantly higher in group A than in group M(P<0.05).Serum E2 and P4 levels were significantly higher in groups A and P than in group M(P<0.05).The expression of ER-αand PGR in the implantation site of endometrial tissue in group A were significantly higher than those in group M(P<0.05).The serum levels of INF-γand IL-2 in group A and group P were significantly lower than those in group M(P<0.05).The serum expression of IL-4,IL-6,IL-10 in group A and group P were significantly higher than those in group M(P<0.05).The endometrium expression of INF-γand IL-2 in group A were significantly lower than those in group M(P<0.05).The endometrium expression of IL-4,IL-6,IL-10 in group A were significantly higher than those in group M(P<0.05).The endometrium expression of INF-γin group P were significantly lower than those in group M(P<0.05).The endometrium expression of IL-6 and IL-10 in group P were significantly higher than those in group M(P<0.05).The mRNA levels of RT-BM1 and these Ly49 receptors(except the Ly49s3 receptor)at the maternal–fetal interface of groups A and P were higher than those of group M(P<0.05).