Acrosomal proteinase and proteinase inhibitor of human spermatozoa.

The acrosomal proteinase of human spermatozoa was characterized and differs from other human proteinases. The enzyme has optimal activity at pH 8.0, is inactive below pH 5.0 or above pH 10.5, requires calcium for maximum activity, hydrolyzes fibrinogen, gelatin, and benzoylarginine ethyl ester, and is inhibited by various synthetic and natural proteinase inhibitors. The molecular weight was estimated to be 30,000. Human spermatozoa also possess a proteinase inhibitor that is similar to one of the proteinase inhibitors from human seminal plasma.

Biological activity assessment of a novel contraceptive antimicrobial agent.

Microbicides are a new category of compounds being developed as a prophylactic approach for the prevention of transmission of sexually transmitted diseases (STDs), including the human immunodeficiency virus (HIV). These are primarily being developed as women-controlled methods, with the target of designing new compounds or formulations that can be used without the knowledge of a male partner. Microbicide screening can be initially based on their hyaluronidase-inhibiting (HI) activity, as this enzyme plays a major role in the sperm and microbe penetration into the substrate. Derivatives of hesperidin, a citrus flavonoid glycoside, have been reported in the literature for their HI effects. Hesperidin was thereby sulphonated under strictly controlled conditions and the active fraction isolated and characterized, based on its HI activity. This derivative was screened for antimicrobial and enzyme-inhibitory activities, specifically for the reproductive tract. Sulphonated hesperidin (SH) was found to completely inhibit the sperm enzymes hyaluronidase, giving an indication toward its contraceptive effects. It was also been found to inhibit various sexually transmitted pathogens, including Chlamydia trachomatis, Neisseria gonorrhoea, HIV, and Herpes Simplex virus type 2 (HSV-2). Its safety assessment was based on its noninterference in sperm motility and its penetration through the cervical mucus, and no effect on the growth of lactobacilli, the normal vaginal flora. It was also found to be nontoxic to the HIV substrate cells (MT2 cells). The study concludes that sulphonated hesperidin can be developed as a potential microbicide for a dual prophylaxis of contraception and transmission of STDs and AIDS.

Synthesis and inhibition of human acrosin and trypsin and acute toxicity of aryl 4-guanidinobenzoates.

The aryl 4-guanidinobenzoate, 4'-nitrophenyl 4-guanidinobenzoate (NPGB), is a potent inhibitor of sperm acrosin, an enzyme with an essential function in the fertilization process. NPGB prevents fertilization in a number of animal species and is a good lead compound for the development of contraceptive agents. In order to assess the efficacy of other aryl 4-guanidinobenzoates as acrosin inhibitors, 24 of these compounds were synthesized. Their inhibitory activity toward human acrosin was determined and compared with their activity toward human pancreatic trypsin in order to assess whether inhibitor sensitivity differed between these similar enzymes. Nine of the inhibitors were synthesized from phenols approved by the FDA for therapeutic use. The acute toxicity of these inhibitors in mice was determined and compared to that of nonoxynol-9, the most commonly used active ingredient in today's vaginal contraceptive preparations. All of the compounds proved to be potent inhibitors of human acrosin although 3 orders of magnitude difference were observed between the most and least effective inhibitors. Little specificity was present in regard to their inhibition of acrosin and trypsin. All the aryl 4-guanidinobenzoates synthesized from FDA-approved phenols were less toxic than nonoxynol-9, and it is concluded that these 4-guanidinobenzoates are of interest for further development and testing as nonhormonal contraceptive agents.

Demonstration of a functional blood-testis barrier to acetaldehyde. Evidence for lack of acetaldehyde effect on ethanol-induced depression of testosterone in vivo.

In vitro studies have shown that acetaldehyde is a more potent inhibitor of testicular steroidogenesis than ethanol. The present study examined the in vivo role of acetaldehyde in ethanol-induced reduction of testosterone by (1) determining the levels of acetaldehyde to which the testes were exposed subsequent to acute ethanol administration to mice; and (2) examining the effect of ethanol on testosterone in animals subsequent to drug pretreatment which decreased or increased ethanol-derived acetaldehyde. Ethanol-induced (3 g/kg) depression of testosterone was dependent upon gonadotropin stimulation. The increase in hCG-induced testosterone was suppressed (P less than 0.01) in ethanol- as compared to saline-treated animals [39.8 +/- 2.6 (S.E.M.) vs 28.1 +/- 2.3 ng/ml]. Pargyline (100 mg/kg) or cyanamide (8.4 mg/kg) increased (P less than 0.05) plasma and testicular acetaldehyde, while having no effect on the testosterone response to ethanol. Similarly, 4-methylpyrazole (25 mg/kg) reduced blood and testicular acetaldehyde to nondetectable levels, while having no effect on testosterone. Testicular acetaldehyde was lower (P less than 0.001) than plasma levels (14 +/- 2 vs 2.0 +/- 0.2 microM). This functional blood-testis barrier to acetaldehyde could be explained by testicular aldehyde dehydrogenases in the mitochondria (Km for acetaldehyde = 1.5 microM) and in the cytosol (Km = 123 microM) whose maximal activities totaled to more than 25-fold greater than that of testicular alcohol dehydrogenase (ADH). ADH was concentrated in the Leydig cells, while aldehyde dehydrogenase was evenly distributed in the testis. Ethanol prevented further hCG-induced rises in testosterone rather than inhibiting testosterone production to below pre-ethanol values. The above data argue against a significant role of acetaldehyde in the in vivo response of testosterone to ethanol. Ethanol appears to impair gonadotropin-testicular receptor interaction in vivo.

Delayed pubertal development of the male reproductive tract associated with chronic ethanol ingestion.

Little is known concerning the sensitivity of the reproductive tract to ethanol as a function of development. The present study was conducted to evaluate the action of chronic ethanol ingestion on sexual maturation of the male. Mice were given free access to liquid diets containing 5% (v/v) ethanol for either 29 or 43 days, starting at age 20 days. Controls were given liquid diets in which isocaloric sucrose replaced the ethanol. Daily diet consumption and peak blood ethanol levels were highest during the first 2 weeks of treatment, dropping thereafter to adult levels of approximately 680 ml/kg body weight and 160 mg/dl respectively. Plasma testosterone levels were depressed by ethanol throughout treatment, the reduction being somewhat greater when measured during week 6 of treatment (average = 74% inhibition) as compared to either week 2 (36%) or week 4 (25%). Average weights of testes, epididymides and seminal vesicles were depressed by 24% (P less than 0.002), 16% (P less than 0.005) and 13% (NS), respectively, after 29 days. Testicular development was also impaired in ethanol-treated animals after 29 days. Tunica albuginea thickness and seminiferous tubule diameter were decreased (by 31%, P less than 0.05; and 16%, P less than 0.01 respectively), whereas desquamation of immature germ cells and inactive tubules were increased (325 and 780% respectively; P less than 0.01). Quality of spermatogenesis was poorer in ethanol-treated animals (P less than 0.05). Also observed were decreased sperm motility (62% inhibition, P less than 0.01) and capacity to fertilize (decreased by 67%, P less than 0.01), and an increase in the incidence of morphologically abnormal spermatozoa (by 163%, P less than 0.001). Semen volume was lower (reduced by 57%, P = 0.05), as was the total number of motile ejaculated spermatozoa (reduced by 81%, P less than 0.05). After 43 days treatment, improvement was noted in all indices of fertility except for the number of motile ejaculated spermatozoa. Significant differences persisted only for dysmorphic spermatozoa and volume and sperm count of electroejaculated semen. These data suggest that ethanol ingestion during pubertal development can delay several aspects of sexual maturation in the male.

Scanning electron microscopy of cervical mucus crystallization.

The crystallization phenomenon of human cervical mucus was studied with the scanning electron microscope. Changes in the conformation of the crystals, crystal conglomerates, and ferning patterns occurred almost daily. During the very early and late stages of the cycle two types of crystals could be found, one columnar and the other flat. At the other times, the columnar crystals were absent and the flat crystals changed their shape to cuboidal. During the follicular phase, these cuboidal crystals were initially located in small groups, then in parallel rows, and finally in three types of ferning patterns, one seemingly typical for the time of ovulation. These patterns reversed during the luteal phase of the cycle. The scanning electron microscope appears to be an extremely useful instrument for the evaluation of these phenomena.

Poly(sodium 4-styrene sulfonate): evaluation of a topical microbicide gel against herpes simplex virus type 2 and Chlamydia trachomatis infections in mice.

OBJECTIVE: To investigate the in vivo activity of poly(sodium 4-styrene sulfonate) (T-PSS) gel formulations as topical microbicides. METHODS: The ability of the gel formulations to reduce the incidence of infection when applied prior to pathogen challenge was examined in mouse models of vaginal herpes simplex type 2 (HSV-2) and Chlamydia trachomatis infection, and rectal HSV-2 infection. RESULTS: In the vaginal HSV-2 challenge studies, 10% T-PSS gel provided significant protection against infection, even when administered 60 min prior to virus challenge (P < 0.0001). Both 5% and 10% T-PSS gel formulations significantly reduced the incidence of upper genital tract C. trachomatis infection in animals treated up to 5 min before challenge (P < 0.001). However, no protection against C. trachomatis infection was seen in animals treated 30 min before challenge. In mice challenged rectally with HSV-2, both the 5% and 10% T-PSS gels significantly reduced infection at 20 s (P < 0.01 for both). However, only the 10% gel provided significant protection when administered 5 min before challenge (P < 0.01). CONCLUSIONS: T-PSS gel formulations have promising in vivo activity as topical microbicides.

Vaginal contraceptive activity of hyaluronidase and cyclooxygenase (prostaglandin synthetase) inhibitors in the rabbit.

Seven inhibitors of the sperm enzyme hyaluronidase were tested at nonspermicidal concentrations for their vaginal contraceptive activity in rabbits. Five of these compounds are marketed antiinflammatory agents and the other two were synthesized. Most of the agents showed contraceptive activity. Of the antiinflammatory agents, phenylbutazone and oxyphenbutazone were particularly potent. Besides being hyaluronidase inhibitors, these compounds are also cyclooxygenase (prostaglandin synthetase) inhibitors; based on other in vitro data, their primary effect on spermatozoa is probably by the inhibition of that enzyme.

Treatment of cervical mucus with lectins: effect on sperm migration.

Treatment of midcycle human cervical mucus with the lectins Ulex europaeus agglutinin I (UE), wheat germ agglutinin (WGA), and peanut agglutinin (PA) did not alter the ability of spermatozoa to enter and migrate through the mucus. These lectins form glycoconjugates by binding to L-fucose (UE), N-acetylglucosamine and sialic acid (WGA), and D-galactose and D-galactose-(1-3)-D-N-acetylgalactosamine (PA), sugars that are present in the carbohydrate side chains of the mucus glycoproteins (mucins). Ricinus communis, with high affinity for D-galactose and N-acetylgalactosamine, did cause a significant decrease in sperm migration but only within the mucus (not at the sperm-mucus interface); this may have been because of an effect on sperm motility. These sugars are thought to be important for the secondary structure of the glycoproteins and for the cross linking between the mucins that produce the rheologic properties of cervical mucus. However, it appears that interference with the sugars by lectin binding does not significantly alter the ability of spermatozoa to migrate through cervical mucus.

Effect of abstinence on sperm acrosin, hypoosmotic swelling, and other semen variables.

OBJECTIVE: To compare the variability of sperm acrosin and hypoosmotic swelling to the more standard semen variables in relationship to controlled periods of sexual abstinence using a defined group of men. DESIGN: Ten men abstained sequentially for 1, 2, 3, 4, 5, and 10 days and produced an ejaculate after each time period. The ejaculate variables measured at each time point were sperm acrosin, hypoosmotic swelling, volume, sperm number, sperm concentration, sperm motility, sperm morphology, pH, and white blood cells (WBCs). Comparisons were performed between the values obtained at each abstinence period. RESULTS: The percentage of hypoosmotically reactive spermatozoa did not vary significantly with the abstinence period. Sperm acrosin remained similar up to 5 days of abstinence but decreased almost twofold after 10 days of abstinence. The sperm volume and concentration increased gradually with the length of abstinence, being approximately twofold higher after 10 days of abstinence than after 1 day of abstinence. The total sperm number increased about fourfold from 1 day of abstinence to 10 days of abstinence. The percent normal sperm forms tended to increase until 5 days of abstinence but decreased after 10 days of abstinence. The WBC count showed only a small increase with longer abstinence periods. The pH remained essentially the same. CONCLUSIONS: The length of abstinence affects the various semen variables differently. An abstinence period of up to 10 days does not alter the hypoosmotic swelling test results. However, the sperm acrosin values decrease after prolonged abstinence so that the abstinence period needs to be taken into consideration when performing this assay.

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.

Postcoital, Vaginal, spermicidal potency of formulations: the Macaca arctoides (stumptailed macaque) as animal model.

The Macaca arctoides (stumptailed macaque) was found to be a good animal model for determining the postcoital spermicidal activity of vaginal preparations. The stumptailed macaque is easy to handle, so formulations can be inserted correctly into the vagina just before coitus. The male mates rapidly, and the entire test can be completed within 5 to 10 minutes, minimizing all extraneous factors other than those inherent to the reproductive tract and the coital act. Data from postcoital breeding experiments were found to be reliable and consistent when results of six primate mating tests for a single dose level of a test formulation were averaged. Dose-response curves can be prepared from these average results, and the relative in vivo effectiveness of the spermicides can be determined. The Sander-Cramer test proved to be a good assay with which to quantitate the in vitro spermicidal potency of formulations. The spermicidal preparations tested immobilized human and primate spermatozoa in vitro to the same extent with the exception of one formulation (possibly due to the vehicle in which the active ingredient was incorporated). The relative spermicidal effectiveness of preparations differs with in vitro and postcoital testing. Because the latter is a more realistic indicator of the contraceptive potency of a formulation, it is recommended that postcoital primate experiments be performed with newly developed vaginal spermicides before extensive animal breeding experiments clinical trials are initiated.

Hemorrhage induced by intrauterine devices: control by local proteinase inhibition.

The intrauterine application of proteinase inhibitors, tranexaminc acid and the pancreatic trypsin inhibitor (Trasylol), reduces or eliminates menorrhagia and intermenstrual bleeding (spotting) produced by an intrauterine device (IUD). A decrease in pain and vaginal (cervical) discharge is also frequently observed. A single application is usually sufficient, more than three never being required. The effect lasts for an average of three cycles. In addition to the clinical use of these agents for the treatment of uterine hemorrhage, the slow release of proteinase inhibitors from an IUD may well be useful in minimizing its side effects without interfering with its contraceptive activity.

PIP: 2 fibrinolytic inhibitors were evaluated in IUD-wearing women complaining of menorrhagia and spotting to determine if direct application into the uterus could reduce such side effects of IUD wear. The agents used were the proteinase inhibitor transexamic acid (AMCA) and a naturally occurring pancreatic trypsin inhibitor Trasylol. Solutions of the antifibrinolytic agents (1 gm/ml AMCA and 19 gm/ml Trasylol) were injected into th eue rine cavity. 10 of the women acted as control. Both agents reduce the length of the menstrual period by about 50% with a significant reduction in pain and vaginal discharge. This effect was usually sustained over several menstrual cycles. Local administration is more advantageous than the oral approach because smaller dosage is required and side effects thus reduced. It is suggested that proteinase inhibitors be incorporated directly into IUDs.

Induction of the human sperm acrosome reaction by human oocytes.

The acrosome reaction of human spermatozoa incubated in the presence or absence of vested human oocytes was investigated. All gametes were obtained from human in vitro fertilization (IVF) cases. Spermatozoa were collected after incubation in insemination medium only and following removal of the oocytes from insemination medium during the IVF procedure. After 16 hours of incubation 18.5% of the spermatozoa in insemination medium alone were acrosome-reacted compared to 31.5% for spermatozoa incubated in medium containing oocytes. The acrosome reaction of spermatozoa incubated with fertilized or unfertilized oocytes was also investigated. The percentage of acrosome reaction did not differ (P greater than 0.05) between the two groups (29.7% in the fertilized cases versus 30.7% in the unfertilized cases). Completion of oocyte nuclear maturation did not affect the proportion of acrosome-reacted spermatozoa observed with unfertilized eggs. A similar (P greater than 0.05) percentage of acrosome reacted spermatozoa were observed regardless of whether the unfertilized oocytes had (29%) or had not (35%) reached metaphase II. These findings indicate the acrosome reaction of human spermatozoa is enhanced in the presence of vested human oocytes. Furthermore, there is no apparent correlation between the percentage of the population of spermatozoa that acrosome react in the medium and the potential of an oocyte for fertilization.

Quality assurance for sperm concentration using latex beads.

OBJECTIVE: To provide a simple, universally applicable method of quality assurance for sperm counting, thereby reducing intercounting chamber variation. DESIGN: By using a known concentration of latex beads, the sperm:bead ratio can be used to calculate the actual sperm count. MAIN OUTCOME MEASURES: The mean sperm and bead counts were determined in both a Spot-lite hemocytometer (Baxter Diagnostics, McGaw Park, IL) and a Makler chamber (Polymedco Inc., Yorktown, NY) from 21 different ejaculates mixed with a known concentration of beads. The hemocytometer chamber was used as the standard counting chamber because it consistently yielded a low variation in sperm count. The adjusted sperm concentration of the Makler chamber was calculated using the following formula [hemocytometer beads]/[Makler beads] x [Makler sperm]. RESULTS: Observed mean +/- SD sperm counts were significantly different between the hemocytometer chamber (110.6 +/- 66.2 x 10(6)/mL) and Makler chamber (173.3 +/- 103.5 x 10(6)/mL). However, calculated Makler chamber sperm counts (118.1 +/- 76.1 x 10(6)/mL) was not statistically different from observed hemocytometer sperm counts. CONCLUSION: This novel approach to sperm counting using a known concentration of latex beads as a reference material can be used to reduce variation in sperm counting between observers, counting chambers, and possibly computerized sperm analyzers.

The zona pellucida-induced acrosome reaction of human spermatozoa is mediated by protein kinases.

OBJECTIVE: To determine if the solubilized human zona pellucida (ZP)-induced acrosome reaction is mediated by protein kinases. DESIGN: Capacitated spermatozoa were incubated with inhibitors of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent kinase (KT5720), Ca(2+)- and phospholipid-dependent kinase (Calphostin C), and cyclic guanosine 3':5'-monophosphate (cGMP)-dependent kinase (KT5823) and then treated with a corresponding kinase stimulator (dibutyryl cAMP, phorbol 12-myristate 13-acetate and dibutyryl cGMP, respectively) to determine the effect on the acrosome reaction. Appropriate controls were performed. Zonae obtained from the unfertilized oocytes of women attending an IVF program were solubilized using acidic NaH2PO4, and the effect of solubilized ZP on the acrosome reaction was tested in dose-response fashion. Comparative studies with solubilized, zona-free oocyte-treated spermatozoa were performed. The effect of the kinase inhibitors on the solubilized ZP-induced acrosome reaction was then determined. RESULTS: No significant stimulation of the acrosome reaction by kinase stimulators occurred when spermatozoa were pretreated with inhibitors of the kinases, in contrast to the controls. Capacitated spermatozoa incubated with 2, 4, and 6 solubilized ZP showed a dose-dependent increase in the acrosome reaction. Solubilized oocytes had no effect on the acrosome reaction. Pretreatment of spermatozoa with kinase inhibitors significantly lowered the acrosome reaction induced by solubilized ZP but not completely. When a "cocktail" of the three inhibitors was used, a significant reduction in the acrosome reaction occurred in comparison with single inhibitor treatment. CONCLUSION: The present data indicate a role for human ZP-induced activation of multiple second messenger pathways, involving kinases A, C, and G in the human sperm acrosome reaction.

Human sperm selection by glass wool filtration and two-layer, discontinuous Percoll gradient centrifugation.

Glass wool filtration and two-layer, discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient centrifugation resulted in an average recovery of 50% to 70% of the progressively motile and about 50% of the hypoosmotic swelling (HOS)-positive spermatozoa. Glass wool filtration tended to be more successful than Percoll centrifugation when the ejaculates were asthenozoospermic or produced a suspect/abnormal HOS test. After selection, the acrosin activity increased approximately two- to threefold, but no significant improvement in the percentage of normal sperm forms occurred. Experiments with mixtures of untreated and frozen-thawed ejaculates confirmed that glass wool filtration is more effective in removing nonmotile and HOS-negative spermatozoa than the two-layer Percoll centrifugation technique when the percentage of these types of spermatozoa in the ejaculate is high. The simplicity of these techniques and the good recovery of apparently viable spermatozoa makes these methods more desirable than other, more complicated techniques or procedures that yield a lower recovery of motile spermatozoa.

Effect of serum proteinase inhibitors on the fertilizing capacity of rabbit spermatozoa.

The in vivo effect of purified human alpha1-antitrypsin and inter-alpha-trypsin inhibitor on the fertilizing ability of capacitated rabbit spermatozoa was investigated and compared with that of the trypsin-kallikrein inhibitor from bovine organs (Trasylol). Only 250 mug of alpha1-antitrypsin/5 X 10(4) sperm in 0.05 ml inhibited fertilization by more than 50%. Lower concentrations of alpha1-antitrypsin (50 mug/5 X 10(4) sperm in 0.05 mo) and inter-alpha-trypsin inhibitor in amounts of 250 mug/5 X 10(4) sperm and less had no significant effect on the fertilization rate. Comparative experiments with Trasylol in conentrations of 50 mug/5 X 10(4) sperm resulted in more than 50% fertilization inhibition, whereas 100 mug/5 X 10(4) sperm decreased the fertilization rate by approximately 90%. The differences in the antifertility effect of these acrosin inhibitors toward capacitated spermatozoa may be due to differences in molecular weight. The effective concentrations required for fertilization inhibition appear to be relatively high under the experimental conditions used.

Cervical mucus penetration test for in vitro assay of vaginal contraceptive agents.

An in vitro penetration test employing bovine cervical mucosa to evaluate vaginal contraceptives is presented. Its usefulness for the evaluation of the potency of a formulation in preventing sperm penetration was assessed by comparison of five commercially available preparations, with the use of both human and primate spermatozoa. The results were compared with those obtained with a spermicidal test (Sander-Cramer method) and in vivo with the use of the primate Macaca arctoides. Analysis of the two in vitro tests and the one in vivo test showed a significant degree of concurrence. The cervical mucus test much more closely reflected in vivo results than the Sander-Cramer test. A recommendation is made regarding the type and order of in vitro and in vivo methods to be used for the development and/or testing of new formulations.

Effect of heterologous seminal plasma on the fertilizing capacity of human spermatozoa as assessed by the zona-free hamster egg test.

The spermatozoa from 8 of 27 men showed a major increase (greater than 15%) in the oocyte penetration rate, the spermatozoa from 7 men showed a major decrease (less than 15%), and no major changes were noted in the other men when the spermatozoa were washed and preincubated with heterologous seminal plasma for 20 minutes before assessing their activity in the zona-free hamster egg assay. No difference was noted in motility or forward progression between the test and control spermatozoa. The increase or decrease in the oocyte penetration rate was consistent for all the ejaculates of a donor; i.e., a donor whose sperm showed an increase in the rate never showed a decrease, and vice versa. Additionally, the oocyte penetration rate of "epididymal-like" spermatozoa, obtained by ejaculation of the first fraction of a semen sample into a large volume of buffer, was enhanced when the spermatozoa were preincubated in their own seminal plasma, obtained from the other fraction of the ejaculate. It is concluded that seminal plasma can have a beneficial influence on the oocyte-penetrating capacity of spermatozoa that is independent of an effect on motility. However, the influence is probably often masked by the presence of antifertility factors in seminal plasma. The penetration-enhancing factor of seminal plasma has a molecular weight of less than 10,000, is quite heat-labile, but is stable at 4 degrees C. The penetration-enhancing factor is somewhat less stable at -20 degrees C and is unstable to lyophilization and reconstitution in H2O. Fertil Steril 40:512, 1983.