Antiretroviral therapy effects on genetic and morphologic end points in lymphocytes and sperm of men with human immunodeficiency virus infection.

Many human immunodeficiency virus (HIV)-infected persons receive prolonged treatment with DNA-reactive antiretroviral drugs. A prospective study was conducted of 26 HIV-infected men who provided samples before treatment and at multiple times after beginning treatment, to investigate effects of antiretrovirals on lymphocyte and sperm chromosomes and semen quality. Several antiretroviral regimens, all including a nucleoside component, were used. Lymphocyte metaphase analysis and sperm fluorescence in situ hybridization were used for cytogenetic studies. Semen analyses included conventional parameters (volume, concentration, viability, motility, and morphology). No significant effects on cytogenetic parameters, semen volume, or sperm concentration were detected. However, there were significant improvements in sperm motility for men with study entry CD4 cell counts >200 cells/mm(3), sperm morphology for men with entry CD4 cell counts < or =200 cells/mm(3), and the percentage of viable sperm in both groups. These findings suggest that nucleoside-containing antiretrovirals administered via recommended protocols do not induce chromosomal changes in lymphocytes or sperm but may produce improvements in semen quality.

Blockade of the alpha(v)beta(3) integrin adversely affects implantation in the mouse.

The role of endometrial and embryonic integrins during implantation remains unresolved although work in animal models and in humans supports their involvement in this process. Temporal and spatial distribution of the alpha(v)beta(3) integrin on both embryo and endometrium in women and mice coincides with the time of initial attachment during implantation. In mice, the endometrial and embryonic alpha(v)beta(3) integrin is present at the time of implantation, as shown by reverse transcription-polymerase chain reaction and immunohistochemistry. In situ hybridization demonstrates the presence of the alpha(v)beta(3) integrin on the subluminal stromal cells of the uterus. Functional blockade of this integrin on the day of implantation by intrauterine injection of neutralizing monoclonal antibodies against alpha(v) or beta(3) integrin subunits, arg-gly-asp (RGD)-containing peptides, or of the disintegrin echistatin, reduced the number of implantation sites compared to controls receiving BSA. These studies demonstrate that, like the human, the murine alpha(v)beta(3) integrin is expressed at the time of implantation in the endometrium and on the blastocyst, and may play a critical role in the cascade of events leading to successful implantation.

Value of estradiol response after human chorionic gonadotropin administration in predicting in vitro fertilization success.

OBJECTIVE: To determine whether the serum E2 response after the administration of exogenous hCG is predictive of outcome during IVF. DESIGN: Prospective, noncomparative cohort. SETTING: Two academic centers and one private-practice IVF program. PATIENT(S): Two hundred twenty-two couples undergoing IVF for infertility arising from ovarian dysfunction, asthenoteratospermia, endometriosis, tubal disease, or unexplained infertility. MAIN OUTCOME MEASURE(S): Implantation, pregnancy, and miscarriage rates were compared in cycles that demonstrated an increase, decrease, or plateau in the serum E2 level on the day after hCG administration. The effects of age, cause of infertility, and maximum E2 value on outcome were evaluated. RESULT(S): Ninety-two cycles resulted in a clinical pregnancy and 130 cycles failed. Of 115 cycles in which the E2 level rose, 42 (37%) resulted in an ongoing pregnancy; among cycles with plateauing E2 responses, 20 of 69 (29%) resulted in a pregnancy. Fifteen of 38 (39%) of cycles exhibiting a drop in serum E2 resulted in an ongoing pregnancy. No statistically significant differences in ongoing pregnancy rates were noted in the increasing, plateauing, or decreasing E2 response groups. CONCLUSION(S): E2 values obtained on the day after hCG administration are not predictive of outcome in women undergoing IVF.

Effect of exogenous gonadotropins on endometrial maturation in oocyte donors.

OBJECTIVE: To determine the effects of controlled ovarian hyperstimulation (COH) on endometrial maturation. DESIGN: Prospective, before and after evaluation of midluteal endometrial biopsies in oocyte donor's spontaneous and subsequent COH cycles. SETTING: Tertiary academic medical center assisted reproductive technologies clinic. PATIENT(S): Nineteen oocyte donors. INTERVENTION(S): Exogenous gonadotropins, endometrial biopsies. MAIN OUTCOME MEASURE(S): Endometrial histology and an immunohistochemical marker of uterine receptivity, the alphavbeta3 vitronectin. RESULT(S): Glandular and stromal dyssynchrony was more common after COH in 16 (80%) of 20 cycles than 6 (30%) of 20 spontaneous cycles (P <.05). Glandular lag was more frequent in COH cycles and unaffected by progesterone administration. The beta3 subunit of the alphavbeta3 vitronectin receptor was present in 9 (45%) of 20 spontaneous and 2 (10%) of 20 COH cycles (P <.05). CONCLUSION(S): Exogenous gonadotropin use in healthy reproductive age women did not result in endometrial evidence of a luteal phase defect. A greater incidence of glandular-stromal dyssynchrony resulted from the use of exogenous gonadotropins. The presence of alphavbeta3 was noted in most endometrial specimens demonstrating in phase glandular maturation. We conclude that endometrial dyssynchrony that results from delayed glandular development most likely represents a normal histologic variant.

Hydrosalpingeal fluid inhibits in-vitro embryonic development in a murine model.

Recent evidence describing a suboptimal clinical outcome in women with hydrosalpinges who undergo in-vitro fertilization (IVF) and embryo transfer suggests a potential deleterious effect of this fluid on in-utero embryo development. Consequently, we evaluated in-vitro mouse embryo development in the presence of hydrosalpingeal fluid (HF) collected from 10 infertile women of reproductive age. Chemical analyses showed both similarities and differences of these fluids to reported values for fluids collected from non-diseased Fallopian tubes. The HF had a significant deleterious effect upon mouse embryo cleavage and development to the expanded and hatched blastocyst stage, although the effect was variable among patients. Dilution of HF to 30% concentration with culture medium failed to negate this effect. This argues against the effect resulting from a relative lack of critical, supportive component(s) in the HF. Additionally, further experiments performed with cultures under an oil overlay significantly reduced the embryotoxicity of the HF. This evidence suggests there may be a lipophilic factor that can impair embryo development. The relatively poor IVF-embryo transfer success in women with proximally patent hydrosalpinges may be explained, at least in part, by reflux of a lipophilic embryotoxic factor(s) into the uterine cavity.

Cigarette smoking at time of in vitro fertilization cycle initiation has negative effect on in vitro fertilization-embryo transfer success rate.

PURPOSE: To assess cigarette smoking of female patients prior to starting in vitro fertilization cycle and possible affect on subsequent in vitro fertilization-embryo transfer outcome. METHODS: Retrospective study involving 340 consecutive patient questionnaires filled out at time of in vitro fertilization program entry. Only cycles resulting in embryo transfer after transvaginal ultrasound directed ovum retrieval (n = 253) were considered. The three patient pregnancy outcomes of not pregnant, spontaneous abortion, and live birth, were cross-referenced with smoking and nonsmoking patients. RESULTS: No significant difference found in overall pregnancy rate per embryo transfer for smokers (35%) vs nonsmokers (31%). However, the abortion rate was significantly higher for the smokers (73%) vs nonsmokers (24%) with a P value < 0.001. CONCLUSIONS: Results suggest preentry in vitro fertilization cycle cigarette smoking has adverse affect on potential pregnancy outcome by increasing spontaneous abortion. Preconception health consultation concerning adverse cigarette smoking effects should be implemented prior to program entry.

In vitro fertilization culture medium surveys. A College of American Pathologists pilot proficiency testing survey.

Two pilot surveys for in vitro fertilization culture medium were conducted by the Reproductive Biology Resource Committee of the College of American Pathologists. The first phase (February 1991) involved seven laboratories, all of them being members of the committee, while the second phase (April 1991) also included other laboratories that voluntarily participated in the survey. Questionnaires accompanied the media and included variables such as conditions of shipment and measurements of pH and osmolarity, along with quality control results obtained from mouse embryo culture studies. Of the two medium samples per shipment, one was adulterated with an embryotoxic substance. The results of these surveys revealed consistency in most laboratories, while some laboratories could be easily identified due to their out-of-range results. These surveys have described the first attempts to develop an intralaboratory testing system for clinical in vitro fertilization laboratories. Further studies will be required to achieve greater consistency among laboratories with respect to implementation of the survey and reporting of results. Additionally, other systems should be evaluated, particularly for those laboratories that use systems other than mouse embryo culture.

Binding of antibodies against antigenic domains of murine lactate dehydrogenase-C4 to human and mouse spermatozoa.

Peptide fragments of lactate dehydrogenase-C4 (LDH-C4) that contain antigenic sequences of the native protein have been identified. The present study describes the binding to murine and human spermatozoa of antibodies that were produced against synthetic peptides containing two of these sequences. Rabbits were immunized with peptides designated MC5-15 and MC211-220, conjugated to diphtheria toxoid (DT). Antisera from these rabbits were tested for binding to washed mouse epididymal sperm or human ejaculated spermatozoa using a solid-phase radioimmunoassay. Antisera bind to mouse sperm in this system at dilutions of 1:64,000. When these antisera are first absorbed with the native LDH-C4 molecule, significant inhibition of binding to sperm results. Antisera to both DT-MC5-15 and DT-MC211-220 bind to human sperm with similar but weaker patterns than seen with mouse sperm. These data indicate that the immune response to synthetic peptides containing antigenic sequences of LDH-C4 includes antibodies that specifically bind to this enzyme on the surface of sperm. In addition, there are shared antigenic sequences between mouse and human LDH-C4, including the MC5-15 and MC211-220 peptides.

Comparison of human acrosin, a trypsin-like sperm proteinase, with human pancreatic trypsin: temperature stability and effect of cations.

Temperature and ion sensitivity of human acrosin (EC 3.4.21.10) was compared to that of human trypsin. With the exception of zinc, no ion tested had significant effects on either enzyme. Zinc behaved as a noncompetitive inhibitor of both enzymes, with inhibition constants of 1.8 and 1.7 mM for acrosin and trypsin respectively. Trypsin was inhibited by the chelators EDTA and EGTA, a specific effect reversed by either calcium or magnesium. EDTA inhibited acrosin in a nonspecific manner, while EGTA was without effect. Unlike acrosin from the other species, human acrosin was unaffected by calcium or the polyamines, spermine and spermidine. Acrosin was sensitive to inhibition by preincubation temperatures above 5 degrees C; trypsin, however, was stable to preincubation temperatures up to 60 degrees C. Hydrolysis of N-alpha-benzoyl-L-arginine ethyl ester was more efficiently catalyzed by trypsin (6800 cal/mol) than by acrosin (9511 cal/mol).

Inhibition of in-vitro fertilization of mouse gametes by proteinase inhibitors.

Kinetic studies were performed to evaluate the interaction of benzamidine (BD), 4-aminobenzamidine (ABD). 4'-nitrophenyl 4-guanidinobenzoate (NPGB), and 4'-methylumbelliferyl 4-guanidinobenzoate (MUGB) with mouse acrosin. The Michaelis constant of mouse acrosin towards alpha-N-benzoyl-L-arginine ethyl ester and the sensitivity of mouse acrosin to inhibitors differed from those reported for other species. NPGB and MUGB were much more active inhibitors of acrosin than BD and ABD. Plots of percentage fertilization versus acrosin inhibitor concentration were generated for all 4 compounds. Linear dose-response curves were obtained and gave ED50 values (50% inhibition of fertilization) of 230 microM for BD, 27 microM for ABD, 35 nM for MUGB, and 13 nM for NPGB. The relative antifertility activity of the compounds paralleled their inhibitory activity towards mouse acrosin, strongly indicating that the inhibition of fertilization is obtained through the inhibition of acrosin. Since the dose-response curves were linear, the mouse in-vitro fertilization system may be useful to screen acrosin inhibitors for their antifertility potency. MUGB should have low toxicity and may have potential as a contraceptive agent.

Antifertility activity of systemically administered proteinase (acrosin) inhibitors.

Low molecular weight, synthetic proteinase inhibitors that inhibit sperm-associated acrosin, were released systemically in female mice at a constant rate from minipumps. The release was timed so that, after mating, the minipumps were depleted of inhibitor before blastocyst implantation took place. Three of the inhibitors: 4-aminobenzamidine (ABD), 4-nitrophenyl-4-guanidino-benzoate (NPGB) and 4-methylumbelliferone-4-guanidinobenzoate (MUGB) caused a 50% decrease in fertility, the last two at very low concentrations. The fourth inhibitor, benzamidine (BD), which is also the weakest inhibitor of mouse acrosin and in vitro fertilization, had no effect. These results show that at least one of the processes leading to fertilization or early blastogenesis, is dependent on proteolytic activity and that the systemic application of proteinase inhibitors inhibits conception. MUGB possessed low toxicity and a high margin of safety, encouraging the development of phenol derivatives of guanidinobenzoic acid as contraceptive agents.

Characterization of a high-molecular-weight form of human acrosin. Comparison with human pancreatic trypsin.

A high-molecular-weight form of acrosin (alpha-acrosin, EC 3.4.21.10) was extracted from spermatozoa obtained from frozen semen and purified over 300-fold. Purification was effected by sequential use of Sephadex G-150, CM-cellulose and DEAE-cellulose chromatography. Properties of human acrosin were compared with those of human pancreatic trypsin. The molecular weight (Mr) of acrosin (70000) was greater than that of trypsin (Mr 21000). Isoelectric points for acrosin (pI = 9.0) and trypsin (pI = 8.2) were also different. alpha-N-Benzoyl-L-arginine ethyl ester was hydrolysed 50% more rapidly by acrosin than by trypsin. Acrosin had similar kcat. values for the hydrolysis of esters with different acylating groups (i.e. benzoyl-L-arginine and p-tosyl-L-arginine esters). In contrast, trypsin had dissimilar kcat. values for the hydrolysis of esters with different acylating groups. Kinetic data argue against deacylation as the rate-limiting step in ester hydrolysis by acrosin. Acrosin was less sensitive than trypsin to inhibition by 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK'), di-isopropyl fluorophosphate and soya-bean trypsin inhibitor. D-Fructose and D-arabinose inhibited acrosin, but had no effect on trypsin. The data indicate that definite differences exist between human acrosin and trypsin.

The role of acrosin in sperm penetration through human cervical mucus.

Enzymes have been implicated in facilitating cervical mucus penetration by spermatozoa. One of these enzymes in the neutral proteinase acrosin, which is associated with the sperm acrosome. To determine the validity of this hypothesis, human spermatozoa were incubated with the following acrosin inhibitors: p-aminobenzamidine (AB), N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and p-nitropheyl-p'-guanidino benzoate (NPGB). An in vitro slide test system was developed which allowed inhibitor-treated and control spermatozoa to be evaluated against the same human cervical mucus sample. At inhibitor concentrations far exceeding those necessary for the inhibition of human acrosin, there was no effect on spermatozoal penetration into or through the mucus. These findings indicate that, in man, acrosin activity is neither necessary nor facilitory to sperm penetration of cervical mucus. Evidence is also presented that demonstrates the superiority of the newly developed double-interface slide test, especially for comparative purposes, over the tests currently in use.