Infertility in mice after unilateral vasectomy

The effects of unilateral vasectomy upon fertility and antisperm antibody production were studied using DBA/1J mice. Thirty-six males underwent either unilateral vasectomy, unilateral orchiectomy, or sham surgery. In vivo effects upon fertility were monitored by matings. Antisperm antibody titers were measured monthly. In vitro fertilization was performed in the presence of serum obtained 4 months postoperatively, and serum testosterone levels were also determined. After 3 months, only 1 male in the vasectomy group induced a pregnancy (1 of 12), while all but 1 of the males in the two control groups induced a pregnancy (20 of 21). The geometric mean antisperm antibody titer was 1:169 in the vasectomy group, while the orchiectomy and sham surgery groups had titers of 1:4 and 1:14, respectively (P less than 0.0001). The percentage of eggs fertilized in vitro in the presence of serum from experimental mice fell from 80% in the two control groups to 40% in the unilateral vasectomy group. Unilateral vasectomy induced infertility in DBA/1J mice and an antisperm antibody response. Sera containing these antibodies inhibited in vitro fertilization. This suggests that infertility after unilateral vasectomy may be immunologically mediated by antisperm antibodies.     

尿激酶型纤溶酶原激活因子在小鼠精-卵接触前通讯和体外受精中作用初探

目的:探讨尿激酶型纤溶酶原激活因子(uPA)在雄性促生育中的作用机制。方法:通过检测精子在毛细管内聚集数量的变化来测试小鼠精子体外对uPA和卵细胞的趋化性,以及将卵细胞分别经uPA、uPA抑制剂(PAI-1)以及抗-uPAR抗体预处理后,检测其趋化诱导精子的能力,来反应uPA在精-卵接触前通讯中的作用。其次观察精子经uPA预处理后其受精能力的变化。结果:精子在递减uPA浓度梯度中的聚集数量明显低于等浓度内的(P<0.05);经uPA预处理的卵细胞其聚集精子的能力显著强于未经处理的卵细胞(P<0.05),而用PAI-1预处理卵细胞后,卵细胞聚集精子的能力显著下降(P<0.05);用抗-uPAR抗体预处理卵细胞并不影响卵细胞对精子的聚集作用;uPA处理后的精子与卵细胞共孵育后其受精卵数目多于未经处理的(P<0.05)。结论:精子在体外对uPA和卵细胞均有趋化性;uPA能增加卵细胞对精子的趋化作用,增进精-卵接触前通讯,促进受精。本实验在一定程度上探讨了uPA促男性生育的机制,并为其在临床上的应用提供一定的理论依据。     

Inhibition of fertilization in the rabbit long after injection of Depo-Provera

Mature female rabbits were given subcutaneous injections of 20 or 50 mg Depo-Provera (Upjohn Company, Kalamazoo, MI) (medroxyprogesterone acetate). Ovulation by mating was inhibited for 40 to 65 days. Although ovulation could be induced by injection of human chorionic gonadotropin, fertilization of eggs failed in all females 15 to 83 days after treatment. When treated and untreated females were inseminated with a definite number of spermatozoa and given human chorionic gonadotropin, fertilized eggs and spermatozoa were found in the oviducts of untreated, but not in the treated, females. Although spermatozoa were found in the uterine horns of treated rabbits, the number was much lower than in the untreated females. It is concluded that long after injection of Depo-Provera, not only was ovulation inhibited, but also fertilization, due to the suppression of sperm transport in the female tracts. The effects of progestins on various aspects of animal reproduction are discussed, stressing the effectiveness and efficiency of their contraception.     

Antifertility effects of antisperm cell-mediated immunity in mice.

C57BL/6 female mice were immunized with allogeneic (DBA/2) sperm in Freund's adjuvant either subcutaneously (s.c.), transcervically into the uterine lumen (i.u.), or with a combination of s.c. and i.u. immunization approaches. Control mice received DBA/2 lymphocytes, human erythrocytes or saline in adjuvant using the same immunization protocols. Immunization with sperm or control cells in adjuvant exclusively by s.c. or i.u. approaches did not affect subsequent fertility, although sperm-injected mice from both protocols had high titers of circulating antisperm antibodies. In contrast, mice that were immunized with sperm in adjuvant by a combination of s.c. and i.u. injections demonstrated significant reductions in fertilization rate and number of viable fetuses and an increased rate of fetal resorption when compared with non-immunized and control-immunized mice. Mice receiving sperm by the s.c./i.u. protocol had high titers of antisperm antibodies and a marked infiltration of T lymphocytes and macrophages into the uterine endometrium. To determine whether cellular immune mechanisms contributed to the infertility effect, T lymphocytes from spleens and pelvic lymph nodes of s.c./i.u. sperm-immunized mice and non-immunized mice were passively transferred to naive syngeneic female recipients which were subsequently mated. The total number of fetuses on day 15 of pregnancy was significantly reduced in mice receiving T-lymphocytes from sperm-immunized mice and a significant increase in fetal resorption sites was also observed. These mice did not have detectable titers of circulating antisperm antibodies, but had a significant infiltration of CD4+ T lymphocytes and macrophages in the uterine epithelium and endometrium. These data indicate that intrauterine antisperm cell-mediated immunity can be induced in mice by a combination of systemic and intrauterine immunizations and provide evidence for the existence of reproductive tract mucosal antisperm cellular immune responses that adversely affect fertility and pregnancy.     

Detection in human sera of antibodies directed against the hamster egg oolemma

Heteroantibodies were demonstrated by indirect immunofluorescence in human sera, which reacted with unfertilized and fertilized hamster eggs. Oolemmal antigens to which these antibodies were directed were distinct from antigen present on the surface of living human spermatozoa. Both species-specific and tissue-specific heteroantibodies were demonstrated by absorption with hamster liver and ovary. An increased degree of heteroantibody binding was noted following penetration of zona-free hamster eggs by human spermatozoa, indicating that an alteration in oolemmal antigen distribution had occurred. No evidence was found, however, that antisperm antibodies in these sera reacted with zona-free hamster eggs following their fertilization.     

Antisperm antibodies and in vitro fertilization

The purpose of this study was to investigate the influence of antisperm antibodies in the male, the female, or both partners on the outcome of in vitro fertilization treatment. The results in terms of ongoing pregnancies in the male and female antibody-positive group were the same as in the antibody-negative group. In the double antibody-positive group two of the three patients became pregnant. When high levels of antisperm antibodies were present on the spermatozoa, the fertilization rate was significantly reduced. In the female positive group no clear relationship between the antibody titer and the fertilization percentage could be detected. Abnormal semen quality was responsible for a much lower fertilization rate than the presence of antibodies. The conclusion of this study is that in vitro fertilization provides an equal chance of conception in couples with antisperm antibodies in comparison with couples with no antibodies if the other semen parameters are normal.     

Passive immunization with antisperm monoclonal antibody ZAP-7 increases preimplantation embryo mortality in the mouse

Objective: To evaluate the effect of VIC-1 and ZAP-7 antihuman sperm monoclonal antibodies on in vivo fertility in the mouse.

Design: A randomized blinded study using a mouse model.

Setting: University-based laboratory.

Animals: B6CBAF1 mice (n = 6 per experimental group).

Intervention(s): Antisperm antibodies were administered intravaginally to female mice before mating. Control mice received no treatment, saline, or nonspecific antibodies. Number and viability of preimplantation embryos were determined by microscopic observation. Mouse sperm, oocytes, and normal preimplantation embryos were used in indirect immunofluorescence assays with antisperm antibodies. The effect of antibody treatment on sperm motility and vitality was evaluated.

Main Outcome Measure(s): Antigen expression, sperm motility and vitality, number and viability of embryos.

Result(s): ZAP-7 antibody recognizes a sperm antigen expressed in zygotes and early preimplantation embryos. Passive immunization with ZAP-7 increases embryo mortality significantly (more than 40% above controls). Passive immunization with VIC-1 has no deleterious effect.

Conclusion(s): ZAP-7 monoclonal antibody disrupts fertilization and embryogenesis in the mouse.     

Relation between antisperm antibodies and the rate of fertilization of human oocytes in vitro

To clarify further the role of antisperm antibodies in in vitro fertilization, the occurrence of antisperm antibodies on ejaculated sperm and in sera was determined by the immunobead binding assay in 67 couples after an unsuccessful in vitro fertilization cycle. Antisperm antibodies in maternal sera were associated with a failure of oocyte fertilization (P <0.02) or with fertilization of only 9–19% of the oocytes (P <0.01) in vitro. Antisperm antibodies were detected in sera from 13 of 24 women (54.2%) where no fertilization occurred, 9 of 14 women (64.3%) where less than 20% of the oocytes fertilized, and 3 of 19 women (15.8%) where greater than 40% of the oocytes fertilized. Antisperm antibodies in these sera were mostly IgG and directed against the sperm tail. Antibodies on the surface of ejaculated motile sperm were also associated with a low (9–19%) fertilization rate (P <0.01). Sperm-bound antibodies were detected in 2 of 24 men (8.3%) where no fertilization occurred, 5 of 14 men (35.7%) where less than 20% of the oocytes fertilized, and 0 of 19 men where fertilization was greater than 40%. Sperm-bound antibodies were mainly IgA and were tail-directed. Antisperm antibodies in sera of males were not related to the rate of fertilization. Antisperm antibodies were detected in female partners of 21 of 46 couples (45.7%) with unexplained infertility, 2 of 12 women (16.7%) with blocked tubes, 4 of 7 women (57.1%) with endometriosis, and 0 of 2 women with adrenal hyperplasia. There was no relation between the fertilization rate and the maternal age, number of oocytes harvested, or semen quality. We conclude that antisperm antibodies are present in sera from a high percentage of women with unexplained infertility and that antibodies reacting with sperm tails may directly interfere with fertilization in vitro or may be a surrogate marker for another factor that interferes with this event.     

Blocking of human fertilization in vitro by sera with sperm-immobilizing antibodies

Serum samples with sperm-immobilizing antibody activity from six women were examined for ability to block sperm-egg interaction by a zona penetration test where human follicular ova matured in vitro were used. Exposure of spermatozoa from a fertile healthy donor to the sera impaired binding to and penetration through the zona pellucida of the spermatozoa completely in five cases and incompletely in one case. Successful fertilization in vitro was achieved by using fetal cord serum instead of autoserum of the patient included in the in vitro fertilization and embryo transfer program. These results suggest that interference with sperm-egg interaction may be an additional mechanism of infertility that is caused by antisperm antibodies.     

Inhibition of sperm penetration through human zona pellucida by antisperm antibodies

An in vitro penetration test using human spermatozoa, sera, and eggs stored in a highly concentrated salt solution was designed for examination of the effect of antisperm antibodies on the process of fertilization. Spermatozoa from a healthy fertile donor incubated in modified Biggers, Whiiten and Whittingham (BWW) medium containing 7.5% antisperm-antibody-negative serum, could penetrate through the zonae pellucidae of the stored eggs, but not when the spermatozoa from the same donor had been incubated in modified BWW medium containing 7.5% antisperm-antibody-positive serum. After the antisperm-antibody-positive serum was absorbed with washed spermatozoa, the sperm penetration was not blocked. Therefore, antisperm antibodies appear to block human sperm penetration through the human zona pellucida.     

Macrophages and infertility: enhancement of human macrophage-mediated sperm killing by antisperm antibodies

The mechanism by which antisperm antibodies inhibit fertility is not completely understood. Macrophages may play a role in mediating infertility by interacting with sperm and destroying gametes. Experiments were conducted evaluating the effect of antisperm antibody on the phagocytosis and lysis of sperm by human peritoneal macrophages in vitro. Sperm from a fertile man treated with sera from normal men and women or medium alone had 5 to 280 molecules of IgG/sperm, as determined by a 125I-labeled anti-human IgG monoclonal antibody assay. By contrast, sperm treated with sera containing antisperm antibodies had 310 to 1240 molecules of IgG/sperm. Peritoneal macrophages harvested from infertile women with tubal/adhesive problems mediated phagocytosis and lysis of 111In-labeled sperm which was enhanced by treatment of the sperm with sera containing antisperm antibodies (39.0% +/- 1.5% versus 76.3% +/- 3.2% phagocytosis, and 3.3% +/- 0.3% versus 23.3% +/- 2.3% lysis of sperm [control versus antibody-treated]). The likelihood of fertilization in couples with antisperm antibody may be determined not only by the antibody but also by the presence of genital tract macrophages capable of destroying the antibody-coated sperm.     

Effect of antisperm antibodies in males on in vitro fertilization

To investigate the effect of antisperm autoantibodies on the process of in vitro fertilization, sperm immobilization test and sperm agglutination test with serum, and direct immunobead test with semen were conducted on the male partners of 101 infertile couples. Fourteen patients were positive in at least one of these three antisperm antibody tests. Six of them were associated with a low fertilization rate (= LFR group, less than 50% of mature oocytes were fertilized), while the other 8 patients had a normal fertilization rate (= NFR group). The spermatozoa in the LFR group were bound to immunobeads of both IgG and IgA classes. In contrast, the spermatozoa in the NFR group were not bound to IgA. The results of the tests with serum were not related to the outcome of fertilization. By rapid dilution and washing of the semen containing antisperm autoantibodies, the percentage of spermatozoa bound to IgG decreased significantly. However, the rate of fertilization was not improved. Six pregnancies were achieved after 13 embryo transfers in the 14 patients. We conclude that the fertilization rate is reduced when spermatozoa are bound to both IgG and IgA antisperm autoantibodies, or to IgA alone. Antisperm autoantibodies do not seem to hamper embryonal development and implantation.     

Successful application of in vitro fertilization and embryo replacement in the treatment of infertile women with sperm immobilizing antibody

Thirteen infertile patients (18 cycles) with sperm immobilizing antibodies were subjected to in vitro fertilization and embryo replacement (IVF-ER) therapy during 12 months from January to December 1985. Four patients became pregnant, two of them delivered healthy babies at term and 2 had abortions at 5 weeks and 12 weeks of gestation, respectively. In the same duration, 24 patients (28 cycles) with tubal factor and 4 patients (5 cycles) with male factor for infertility were subjected to IVF-ER, and two patients with tubal factor became pregnant and delivered healthy babies at term. In the patients with immunological factor, fertilization and cleavage rates per mature oocyte were 85.9% (55 fertilized/64 oocytes) and 81.3% (52 cleaved/64 oocytes) respectively, while the fertilization rates in patients with tubal factor and male factor were 70.5% (62 fertilized/88 oocytes) and 50.0% (6 fertilized/12 oocytes) respectively and all fertilized eggs in these patients developed to the cleavage stage. Thus fertilization and cleavage rates for mature oocytes from the patients with the immunological factor were slightly better than those with the tubal factor and much better than those with the male factor. The antisperm antibody titers (SI50) in sera determined by the quantitative sperm immobilization test ranged from 20 to 243 units while those in follicular fluids ranged from 21 to 160 units in the patients with the immunological factor. The follicular sperm immobilizing antibodies could be detected in any patient who had the antibodies in the serum. Immunoglobulin (IgG, IgA, IgM) concentrations in follicular fluids were not significantly different from each other in the patients with immunological, tubal and male factor for infertility.     

Monoclonal antibodies identify Fc gamma receptors on unfertilized human oocytes but not spermatozoa.

Oolemmal Fc receptors have previously been shown to play a role in the promotion of adhesion by antibody labeled human spermatozoa to zona-free hamster eggs. In this work, we demonstrated the presence of Fc gamma RI, Fc gamma RII and Fc gamma RIII on the oolemma of unfertilized human oocytes by means of monoclonal antibodies directed against these receptors, detected both by immunobead rosetting and indirect immunofluorescence. These receptors were also functionally active in that they were able to bind human aggregated IgG, human IgG-Fc, mouse IgG1 and IgG2a. While the presence of oolemmal IgG-Fc receptors might play a role in reproductive failure, by their promotion of polyspermic fertilization, in cases where antisperm antibodies bound to the spermatozoan surface, their role in the normal physiology of fertilization or in other events unrelated to sperm incorporation remains to be determined. In contrast, Fc gamma receptors were not present on human spermatozoa, irrespective of their functional state (fresh ejaculated, capacitated or acrosome reacted).     

Inhibitory effects of monoclonal sperm antibodies on the fertilization of mouse oocytes in vitro and in vivo

The inhibitory effects of monoclonal antibodies on the fertilization of mouse oocytes were evaluated in vitro and in vivo. Among the 40 sperm-specific monoclonal antibodies that had been examined, nine showed significant inhibition of the fertilization of mouse oocytes in vitro. MS 204 was shown to cause a high incidence of penetration of the zona pellucida of mouse eggs by multiple sperm, when the sperm concentration for insemination exceeds 1 X 10(5)/ml. This antibody prevented further penetration of the vitelline membrane by sperm. On the other hand, sperm penetration to zona was inhibited in the presence of MS 207. However, neither MS 204 nor MS 207 caused significant inhibition of penetration of zona-free mouse or hamster eggs by sperm. MS 204 and MS 207 were also found to inhibit the fertilization of mouse oocytes in vivo and embryo development in vitro, when superovulated mice were injected intraperitoneally with given doses of antibodies prior to the mating. Further in vitro culture of the recovered 2-cell oocytes revealed little or no further embryo development beyond two- to four-cell stages. In the controls, greater than 80% of the retrieved oocytes were fertilized and successively developed to the blastocyst stages in vitro when the ascites fluid from NS-1 cells was administered. Two of the monoclonal antibodies generated against human sperm antigens, HS 11 and HS 63, were shown to cross-react specifically with mouse sperm acrosomal antigens and also inhibited the fertilization of mouse oocytes in vitro and in vivo. The results of this study suggest that some monoclonal antibodies to sperm acrosomal antigens exhibit strongly inhibitory effects on the in vitro and in vivo fertilization of mouse oocytes as well as subsequent development of early embryos. As a comparative control, rabbit antisera against sperm-specific enzymes, lactate dehydrogenase-X and 3-phosphoglycerate kinase-2, showed little or no inhibition on the fertilization of mouse oocytes in vitro or in vivo or the subsequent embryo development.     

The detection in human sera of antisperm antibodies reactive with FA-1, an evolutionarily conserved antigen, and with murine spermatozoa

Evolutionarily conserved antigens are present on spermatozoa of several mammalian species. We tested sera from infertile men and women containing antisperm antibodies (ASAs) for their reactivity with FA-1, an antigen known to be present on murine and human spermatozoa. Fifty percent of male sera and 63% of female sera contained anti-FA-1 antibodies, as judged by enzyme linked immunosorbent assay (ELISA). Fourteen percent of male sera and 50% of female sera were also shown to possess ASAs reactive with living mouse spermatozoa, and murine in vitro fertilization was inhibited by human antibodies. These results suggest that the transfer of immunoglobulins from human sera to spermatozoa of other species may provide a model to study how ASAs effect sperm function.     

Studies on biological actions of sperm immobilizing antibody on human fertilization in vitro

Effects of sperm immobilizing antibodies on sperm penetration through human zonae pellucidae have been studied. Exposure of human spermatozoa obtained from fertile donors to seven serum samples with sperm immobilizing antibody impaired sperm penetration completely in six cases and incompletely in one case. During the course of treatment of a patient with circulating sperm immobilizing antibody by means of an in vitro fertilization and embryo transfer program, it was found that fertilization was completely blocked in the presence of the patient's serum, but three matured ova fertilized successfully when umbilical cord serum was used instead of autoserum from the patient. Furthermore, when spermatozoa were exposed to an IgG fraction of sera containing sperm immobilizing antibody, sperm binding and penetration were markedly inhibited. The spermatozoa, preincubated with sperm immobilizing antibody, showed penetrability across the zona pellucida. However, exposure of possibly capacitated sperm to the antibody completely blocked sperm binding to and penetration through the zona pellucida. These results suggest that sperm immobilizing antibodies cause infertility by preventina sperm binding to and penetration through the zona pellucida, possibly by interfering with the step of fertilization beyond sperm capacitation.     

Reduction of fertility in female rabbits and mice actively immunized with a germ cell antigen (GA-1) from the rabbit

Female rabbits and mice were actively immunized against germ cell antigen (GA-1) of 63 kDa molecular mass isolated from rabbit sperm and testis. There was a significant (P less than 0.05) reduction of fertility in rabbits actively immunized with GA-1 as compared to controls, as seen by the percentage of 9-day implants/corpora lutea ratio (GA-1, 36.3%; controls, 85.7%). In mice, there was again a significant (P less than 0.01) reduction in fertility as seen by mean 7-9 day implants +/- S.D. per mated mouse actively immunized with GA-1 whether through the intraperitoneal route (GA-1, 1.2 +/- 1.6; controls, 8.0 +/- 3.4) or through the subcutaneous/intramuscular route (GA-1, 3.8 +/- 3.4; controls, 10.1 +/- 3.9). The antisera from these actively immunized animals were negative for sperm agglutinating and immobilizing antibodies. In the Western blot enzyme-immunobinding procedure, the antisera showed specific binding to a single protein of 63 kDa. The incidence of fertilization of eggs recovered from rabbits inseminated with anti-GA-1 antibodies-treated sperm was not significantly different from control rabbits. The percentage of fertilized eggs obtained from rabbits inseminated with anti-GA-1 antibodies-treated sperm that reached the blastocyst stage upon in vitro incubation, however, was significantly less than that for embryos obtained from rabbits inseminated with control serum-treated sperm. Incubation of normal fertilized eggs in vitro with the antibodies did not affect development. Neither antiserum nor immune uterine fluid reacted with 4-day blastocysts in the indirect immunofluorescence technique. It is concluded that active immunization with GA-1 results in post-fertilization reduction of fertility in rabbits and mice by inhibiting early embryonic development.     

Immunologic infertility

In general, the management of antibody-mediated infertility has been plagued by misdiagnosis due to the choice of assay system, therapies that may be associated with major side effects, and a failure to learn from the models of other antibody-mediated diseases. An important consideration in diagnosing antibody-mediated infertility is to use an immunoglobulin-specific technique that employs intact or living spermatozoa. Once this is done, a search should be made for what functional deficits in reproduction are associated with the presence of the antisperm antibodies. This would include postcoital testing, tests for ovum penetration (human sperm/hamster egg penetration assay), and tests for the ability of the sperm in the presence of antibody to undergo the acrosome reaction. Once the extent of the reproductive deficit is known, appropriate therapy can then be suggested. I believe that a "case" must be built to prove that antibody-mediated fertility actually exists in a particular couple, even in the presence of a positive test that is known to be highly specific. Sperm antibodies can attach to the sperm's surface without detrimentally affecting reproductive function. In a similar vein, low levels of sperm antibodies could give a positive test result, but the level may be insufficient to be a major detriment to fertility. It is hoped that in the near future isolation of specific sperm antigens will be used to identify antibodies against antigens critical to reproductive function. It may be possible to determine the minimal amount of antisperm antibody that is necessary to disturb each individual step in reproductive function. If a patient is found to have sperm antibodies whose quantity exceeds this amount, then the patient can be appropriately labeled as having antibody-mediated infertility. The array of therapies available for the couple's unique fertility problem(s) can then be described to the patient, and an appropriate therapeutic choice made.     

Failure of intrauterine insemination in male immunological infertility in cases in which all spermatozoa are antibody-coated.

OBJECTIVE: To determine if the overcoming of the cervical mucus barrier removes the interference of sperm-bound antibodies with fertility. DESIGN: Prospective case series. SETTINGS: University-based intrauterine insemination (IUI) homologous program. PATIENTS: Nineteen patients with all spermatozoa in the ejaculate coated by antisperm antibodies. As control group, 86 consecutive patients without antisperm antibodies, treated for oligoasthenozoospermia or mucus hostility. INTERVENTIONS: Intrauterine inseminations (at least 3 attempts per couple). MAIN OUTCOME MEASURES: The outcome of IUIs, demographic, and seminal parameters were compared between the two groups. RESULTS: No pregnancy occurred in the couples with male immunological infertility, treated by 110 IUIs. Twenty-three pregnancies occurred in 22 (25.6%) of the control group couples who were treated by 411 IUIs. In the group of patients without antisperm antibodies, we demonstrated that the pregnancy rate (PR)/couple in oligoasthenozoospermia without teratozoospermia was similar to that achieved in normozoospermia (35% versus 38.9%), whereas it was significantly affected by teratozoospermia (3.6%). Only three patients with antisperm antibodies had teratozoospermia. Comparing the PR per couple and per cycle between the two groups of patients (with and without antisperm antibodies), excluding the patients with teratozoospermia, significant differences resulted (P less than 0.005 and P less than 0.005, respectively). The motile sperm count was not significantly different between the two groups, which also resulted to be homogeneous for demographic data. Moreover, the motile sperm count was not different between the patients with and without antisperm antibodies, who had successful IUI. CONCLUSIONS: The analysis of this trial suggests that the failure of IUI in the treatment of male immunological infertility is imputable to antisperm antibodies when they involve all spermatozoa, regardless of semen quality.