Effects of intrauterine infusion of seminal plasma at artificial insemination on fertility of lactating Holstein cows.

An inflammatory response is induced in the reproductive tract by deposition of semen during natural mating. This response might facilitate establishment and maintenance of pregnancy and alter the phenotype of the offspring by modifying the microenvironment of the reproductive tract. Here, we hypothesized that intrauterine infusion of 0.5 mL of seminal plasma at the time of artificial insemination (AI) in first-service lactating Holstein cows will improve pregnancy success after insemination. Cows were inseminated (511 primiparous cows inseminated with X-sorted semen, 554 multiparous cows inseminated with X-sorted semen, and 627 multiparous cows inseminated with conventional semen) using the Double-Ovsynch protocol. Cows were randomly assigned to receive intrauterine infusion of either 0.5 mL of seminal plasma or saline immediately after AI. There was no overall effect of seminal plasma infusion on the percentage of inseminated cows diagnosed pregnant at d 32 or 60 after AI, pregnancy loss, or percent of inseminated cows calving. If cows were inseminated with conventional semen, seminal plasma reduced pregnancies at d 32 and tended to reduce calvings. There was no effect of seminal plasma if cows were inseminated with X-sorted semen. Seminal plasma infusion increased the birth weight of heifer calves born using X-sorted semen but not conventional semen. These results do not support a beneficial effect of seminal plasma on pregnancy success after AI, but exposure to seminal plasma may program fetal development to affect phenotype at birth.

Markers of the uterine innate immune response of the mare.

Reproductive efficiency in mares is low and persistent mating-induced endometritis (PMIE) is an important cause of subfertility. Mating-induced endometritis (MIE) an obligate precursor to PMIE, is a ubiquitous, transient inflammatory response to the presence of sperm, seminal components and pathogens. However, the specific inflammatory pathways that derive from MIE and that may also be precursors to PMIE are not clear. The ability to identify and measure robust, repeatable markers of inflammation integral to MIE may be key to understanding the progression to PMIE. The aim of the study was to (i) refine a protocol for inducing MIE and in doing so test a range of cellular and molecular parameters as valid markers of MIE to facilitate future studies of mares susceptible to PMIE (ii) concurrently identify those parameters with potential as inflammatory indicators during MIE to inform and enhance early treatment regimens in practice. Mating-induced endometritis was induced in pony mares using a stringent protocol; mares were treated intrauterine with frozen/thawed semen (n = 5; FTS) or frozen/thawed extender (n = 6: FTEx). The parameters tested were measured before treatment were compared to samples collected at strategic time points after treatment: uterine cytology using cytological (at 8, 16, 24, 48 and 72 h after treatment) or histological analysis (at 24 and 72 h); uterine bacteriology (at 24 and 72 h); secretion of prostaglandin F(2alpha) (PGF(2alpha); at 8, 16, 24, 48 and 72 h); peripheral concentrations of serum amyloid A (SAA; at 24h); endometrial mRNA gene expression, focussing upon IL8 and TLR4, as examples of genes pertinent to inflammation (at 24 h). Uterine neutrophil cell numbers in both treatment groups increased at 8 (P < 0.001), 16 (P < 0.01) and 24 (P < 0.01) h after insemination, indicative of MIE and distinguished between different treatments because neutrophil numbers were greater from FTS mares than FTEx mares 8h after challenge. Uterine neutrophil cell numbers, assessed by histology, increased (P < 0.001) 24 and 72 h after treatment. Prostaglandin F(2alpha) concentrations increased (P < 0.05) 16 h after treatments, while SAA concentrations and bacterial growth scores were not significantly different after treatment. Endometrium from pony mares expressed mRNA for IL8 and TLR4 but expression was not altered after insemination. The protocol induced MIE, as confirmed by uterine cytology and maybe used hereafter as a repeatable and robust method for studying immune mechanisms that underlie MIE and so may aid the understanding of progression to persistent inflammation. It can be concluded that of the range of parameters tested, neutrophil cell numbers by cytological analysis and PGF(2alpha) were regarded as the most accurate markers of inflammation during MIE and important for use in practice.

Changes in the localization of antigen presenting cells and T cells in the utero-vaginal junction after repeated artificial insemination in laying hens.

The goal of our present study was to observe whether the populations of antigen presenting cells (Ia+ cells) and T cell subsets (CD4+ and CD8+ T cells) change in the utero-vaginal junction (UVJ) of Rhode Island Red laying hens that showed dramatic declines in fertility after repeated artificial insemination (AI). Rhode Island Red laying hens were divided into two groups: a virgin group (R-V) and artificial inseminated group (R-AI), which was exposed to weekly AI for a period of 3 mo. Undiluted fresh semen collected from healthy Tosa-Jidori roosters, a native Japanese breed maintained in Kochi Prefecture, was used for AI. The UVJ tissues were processed for frozen sections, and Ia+ cells and CD4+ and CD8+ T cells were identified by immunohistochemistry. The Ia+ cells and CD4+ and CD8+ T cells were observed in the stroma and mucosal epithelium of UVJ in both the R-AI and R-V birds. The frequencies of them in the stroma were significantly higher in R-AI than R-V. The higher frequency of Ia+ cells in the UVJ of R-AI group indicated a greater potential capability for antigen presentation to CD4+ cells. The significant increase in CD8+ and CD4+ T cells in the UVJ of R-AI birds might be the result of a homing process of lymphocytes, which may affect sperm survivability and fertility.

Uterine immune reaction and reproductive performance of sows inseminated with extended semen and infused with pooled whole dead semen.

The objective of this study was to investigate the effect of infusing whole dead semen (WDS) after AI with diluted commercial semen on uterine inflammatory reaction and embryonic survival rate in gilts. Sixty Yorkshire-Landrace gilts were assigned at their second estrus to one of the following AI treatments: 1) commercial semen adjusted to 1 x 10(9) sperm cells (S1) per dose, followed by an infusion of 80 mL of WDS (S1-WDS); 2) S1 followed by an infusion of 80 mL of Beltsville Thawing Solution (S1-BTS); 3) commercial semen adjusted to 3 x 10(9) sperm cells (S3) per dose, followed by an infusion of 80 mL of BTS (S3-BTS); and 4) a negative control group, in which gilts received two infusions of 80 mL of BTS (BTS). Two days after the first AI, eight gilts from Groups 1, 2, and 4 were slaughtered and reproductive tracts were collected. One horn was cut open longitudinally along the antimesometrial aspect and endometrial samples were taken and immediately frozen for analysis of messenger RNA (mRNA) abundance for inflammatory cytokines and growth factors. The other horn was flushed with 20 mL of PBS, and the contents of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) were determined by ELISA. On d 25 after AI, gilts from Groups 1, 2, and 3 were slaughtered and their reproductive tracts were collected to evaluate the number of fetuses and corpora lutea. On d 2 after the first AI, only TGF-beta1 was detected in the flush of all gilts, and no difference was observed between S1-WDS, S1-BTS, and BTS gilts. Endometrial levels of IFN-gamma and interleukin (IL)-6 mRNA were marked in all gilts, but they were not affected by the AI treatments, whereas the mRNA abundances for IL-1 and IL-2 were negligible. Infusions of WDS or BTS after a fertile AI did not affect IGF-I, IGF-I receptor, or IGF-II mRNA levels compared with gilts infused with BTS only, whereas the mRNA abundance for the IGF-II receptor was decreased (P < 0.05) in WDS-infused gilts. In gilts inseminated with S1 doses, infusion of WDS did not affect the number of live embryos. Although infusions of WDS did not affect the mRNA level and secretion of the cytokines measured and did not improve embryonic survival rates, further studies are needed to better understand the influence of semen composition on the uterine response after mating.

Endometrial IL-1beta, IL-6 and TNF-alpha, mRNA expression in mares resistant or susceptible to post-breeding endometritis. Effects of estrous cycle, artificial insemination and immunomodulation.

Endometrial mRNA expression of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) was assessed in mares resistant (RM) or susceptible (SM) to persistent post-breeding endometritis (PPBE). Eight RM and eight SM, were selected based on reproductive records and functional tests out of a herd of 2,000 light cross-type mares. Three experiments were done to study transcription patterns in (i) basal conditions; (ii) after artificial insemination (AI); and (iii) after administration of an immunomodulator at time of artificial insemination. Endometrial biopsies were taken during consecutive cycles: (i) at estrus, when follicles reached 35 mm and at diestrus (7 +/- 1 days after ovulation); (ii) at 24 h post-AI, with dead semen (estrus) and in diestrus; (iii) at 24 h after treatment with a Mycobacterium phlei cell-wall extract (MCWE) preparation and AI (with dead semen), and at diestrus. mRNA expression was quantitated by real time PCR. Under basal conditions, SM had significantly higher mRNA expression of all cytokines in estrus and of IL-1beta and TNF-alpha in diestrus, compared to RM. After AI, there were no differences between RM and SM in estrus; however, mRNA expression for all three pro-inflammatory cytokines was higher than under basal conditions. In diestrus, RM showed significantly lower IL-1beta and TNF-alpha mRNA expression than SM. When MCWE was administered at time of AI, no differences between cytokine induction from RM and SM were found. Globally, mRNA expression for all three cytokines correlated well among themselves when expression was high. The present study showed that (i) in basal conditions RM had lower mRNA expression of pro-inflammatory cytokines than SM with no effect of estrous cycle; (ii) AI upregulated mRNA expression for all three cytokines in both RM and SM, with persistance in diestrus in the latter; (iii) treatment with MCWE at time of AI down-regulated mRNA expression of IL-1 with significant effects in SM which behaved like RM. Immunomodulation with MCWE could be of help in restoring homeostatic local inflammatory mechanisms, thus assisting in the prophylaxis of post-breeding endometritis in mares.

Characterization of uterine leukocyte infiltration in gilts after artificial insemination.

The objective of this study was to characterize the uterine leukocyte influx after artificial insemination (AI). After detection of oestrus with a boar at intervals of 1.5 h, seventy-two gilts were randomly assigned to a 2 x 3 x 4 factorial arrangement. AI was performed with 100 ml extended semen containing 5 x 10(9) spermatozoa (semen; n = 36) or 100 ml VSP semen extender (extender; n = 36) at one of three times after detection of oestrus: 12, 24 or 36 h (n = 24/time). The uterus was lavaged at 6, 12, 18 or 24 h (n = 18/time) after AI to determine the total number of uterine leukocytes. In addition, uterine lavage was performed on nine untreated gilts immediately after the detection of oestrus to establish a baseline number of leukocytes. The leukocyte response in all samples consisted predominately (92-99%) of polymorphonuclear neutrophilic granulocytes (PMNs). The mean number of PMNs recovered from the uteri of gilts treated with semen was greater than in gilts treated with extender and in untreated gilts (P < 0.01). The greatest number of PMNs in semen-treated gilts was found 12 h after AI (P < 0.01), and this number was sustained for 24 h. In contrast, the number of uterine PMNs recovered from extender-treated gilts reached a peak at 6 h and had declined by 12 h after AI (P < 0.05). It was concluded that an extensive influx of PMNs into the uterus is a normal sequence to AI. The consequences and importance of semen-induced uterine leukocytosis needs further investigation.

Cervical mucus anti-sperm antibodies: treatment with intrauterine insemination.

PROBLEM: To determine (1) the incidence of cervical mucus anti-sperm antibodies in infertile women, and (2) the results of treatment by intrauterine insemination. METHOD: Cervical mucus was collected the morning after urinary LH surge occurred from 153 consecutive women being treated for unexplained infertility with intrauterine insemination. Immunobead testing for IgG, IgA, IgA1, and IgA2 was performed with only actively motile sperm being counted. RESULTS: Overall, 23/153 (15.0%) of cervical mucus samples were positive for anti-sperm antibodies: 9/23 (39.1%) were only IgA-positive (62% IgA1-positive, 38% IgA2-positive), 11/23 (47.8%) were only IgG-positive, and 3/23 (13.0%) were positive for both IgA and IgG. Insemination resulted in a pregnancy in 6/23 (26.1%) of women with cervical mucus anti-sperm antibodies after 1-3 cycles. CONCLUSIONS: Testing for cervical mucus anti-sperm antibodies should be performed in cases of "unexplained" infertility, and intrauterine insemination may be an effective treatment, resulting in pregnancies in over one-fourth of couples.

Effect of immunization with lutropin-receptor on the ovarian function of rabbits.

Three New Zealand white adult female rabbits, designated as A, B, and C, were immunized to produce lutropin receptor antibodies. Antisera inhibited the binding of 125I-hCG to the lutropin receptor and the production of testosterone by hCG stimulated rat Leydig cells. During the study period of, approximately 10 months after the last immunization, rabbits did not ovulate in response to an injection of 75 IU of human chorionic gonadotropin or mating as revealed by laparotomy. As the antibody titers declined, induction of ovulation and laparotomy revealed restoration of ovulation and corpus luteum formation. However, no pregnancy occurred when rabbits A and B were mated and artificially inseminated. These observations indicate that lutropin receptor antibodies can cause infertility in female rabbits.

The frequency of antisperm antibodies in the cervical mucus of women with poor postcoital tests and their effect on pregnancy rates.

PROBLEM: To determine the impact of the presence of antisperm antibodies (ASAs) in the cervical mucus of female partners in couples with unexplained poor postcoital tests (PCT). Furthermore, the efficacy of intrauterine insemination (IUI) in these same patients was determined by pregnancy rates (PRs). METHOD: Pregnancy rates following IUI in patients with infertility and poor postcoital tests, whether the cervical mucus was positive or negative for ASAs, were evaluated. RESULTS: The 6-month PRs were similar in the ASA negative (40.5%) versus the positive (42.4%) group. CONCLUSIONS: It appears that the antifertility effect of ASA may be mainly the immobilization of sperm in the cervical mucus, and thus, performing IUI may effectively correct the problem.

Rapid transport of seminal immunoglobulin to distal parts of the female reproductive tract in rabbits.

Bucks immunized with the bacteriophage-cloning vector fd-tet display strong humoral seminal immunity of 1.5 x 10(6) fd-tet neutralizing units (nu) ml-1. We used this model to study the distribution of seminal immunoglobulin in the female reproductive tract after copulation and artificial insemination. In contrast to artificial insemination, significant (P < 0.01) amounts (147 nu ml-1, i.e. about 1.25 x 10(-4) input ejaculate) of neutralizing activity reached the uterus within 20 min of a single copulation, which is evidence for rapid transport of seminal plasma to distal parts of the tract. Oviducts were also quickly impregnated with antibodies to fd-tet, which persist in the distal compartments for at least 24 h.

Effect of antisperm antibodies on postcoital results and effect of intrauterine insemination on pregnancy outcome.

The effect of antisperm antibodies (ASA) in males was determined in 59 men using the direct immunobead test (IBT). Postcoital tests were evaluated in couples for whom all female factors appeared to be corrected. Pregnancy rates in 6 months were compared in couples with good postcoital tests vs. those with poor results; the latter group was treated with timed intrauterine insemination (IUI). Thirty-one percent of males with positive ASA (greater than or equal to 50%) had normal postcoital tests and all four achieved pregnancies. Fifty-six percent of men with positive ASA and poor postcoital scores achieved pregnancies following IUI therapy of their wives in 6 months; 83% of couples with normal postcoital tests achieved pregnancies as did couples treated with IUI when the postcoital was poor but the male ASA negative.

Does intraperitoneal insemination in the absence of prior sensitization carry with it a risk of subsequent immunity to sperm?

To evaluate the occurrence of antisperm antibodies in women, with no prior sensitization, 112 couples undergoing intraperitoneal insemination were tested for serum antisperm antibodies with the sperm immobilization test (SIT) and the immunobead test (IBT). A serum sample was taken from each of the 112 patients immediately before the first intraperitoneal insemination. Another sample was taken from 58 patients who underwent a second insemination procedure. In 16 of the 58 patients the IBT results were positive for one or more immunoglobulin classes. Five patients showed positive SITs. In 7 out of these 16 subjects (12%) the antibodies were bound to the head and to the shaft of the sperm tail. Five of the six patients submitted to a third intraperitoneal insemination procedure showed unchanged SIT values and IBT binding percentages. In one subject, SIT (6 months after the third insemination) became negative. Antibody production may be either a transient response to massive antigen stimulation or the first step toward systemic immunity.

Intrauterine inseminations with washed human spermatozoa does not induce formation of antisperm antibodies.

In this study we investigated the possible development of serum antisperm antibodies in women receiving repeated IUI. Patients acted as its own control and were evaluated before and after various (1 to 15) IUI cycles using three different assays for antisperm antibodies. It was found that only 2 out of 41 women developed antisperm antibodies. We concluded that exposure of the upper reproductive tract to washed spermatozoa during repeated IUI with partners' sperm does not significantly stimulate the appearance of serum antisperm antibodies.

Normal fertilization in men with high antibody sperm binding by the addition of sufficient unbound sperm in vitro.

A high incidence of fertilization failure has been reported in men with over 70% of their sperm bound with isoantibodies. In three men with greater than 80% antisperm antibody binding with IgG and IgA immunoglobulins, a normal rate of fertilization (29/46 oocytes; 63%) was achieved by adding a sufficient number of motile sperm to provide at least 50,000 unbound sperm per oocyte. This method appears to be simpler and more effective than attempting to separate unbound sperm in vitro.

Sperm antibodies after intraperitoneal insemination of sperm: a preliminary report.

To test the hypothesis that intraperitoneal insemination of sperm induces the expression of anti-sperm antibodies a prospective study was designed. Fifteen women undergoing intraperitoneal insemination (with or without oocyte transfer) were studied with 11 women having evaluation of anti-sperm antibodies. Sperm antibodies were detected by the immunobead test prior to intraperitoneal insemination and after each treatment cycle. Two criteria were used to assess positivity: the first based upon negative controls and the second based upon the evaluation of 20 fertile control couples. Using the first criteria, of 11 of the women undergoing IPI for the first time, 7 were initially negative and 4 were initially positive for at least 1 isotype. After treatment, 3 additional patients were positive (for a total of 7) and 4 patients remained negative. This alteration in sperm antibody frequency was not different (P = .4) as determined by the Fisher's Exact Test. Four of the 11 patients underwent a second cycle of IPI. All 4 patients were negative prior to the first treatment and 3 were negative prior to the second treatment. Subsequent to the second exposure, all 4 of these women were positive for at least 1 isotype. This shift in frequency distribution after 2 cycles was significant (P = .01). The frequency of antisperm antibodies for the same 11 patients was evaluated by using fertile control values as the basis of positivity. Two patients (18%) were positive for anti-sperm antibodies prior to intraperitoneal insemination. There was no change in the frequency of positivity after 1 cycle of IPI.(ABSTRACT TRUNCATED AT 250 WORDS)