Recommendations on the pharmacological treatment of epithelial ovarian cancer in Mexico.

Ovarian cancer is the third most frequent gynaecological malignancy worldwide and in Mexico, with a high mortality rate, due to that in many cases its diagnosis is made in advanced stages. Prognosis is important for determining the subtype and the degree of evolution. During lasts years, the management of ovarian cancer has undergone an important evolution with the incorporation of new therapeutic options, which in turn represent an increase in the survival of these patients. We present recommendations for the management of ovarian cancer developed by an expert panel Mexican based on available evidence so far and the characteristics of health care in the country.

El cáncer de ovario es la tercera neoplasia maligna ginecológica más frecuente globalmente y también en México, con una elevada tasa de mortalidad debido a que en muchos casos su diagnóstico se realiza en etapas avanzadas. Para establecer su pronóstico es importante la determinación del subtipo y del grado de evolución. En los últimos años, el manejo del cáncer de ovario ha sufrido una importante evolución con la incorporación de nuevas opciones terapéuticas, que a su vez representan un incremento en la supervivencia de estas pacientes. Se presentan las recomendaciones para el manejo del cáncer de ovario elaboradas por un panel de expertos mexicanos basadas en la evidencia disponible hasta el momento y en las características de la atención sanitaria del país.

Recomendaciones sobre el tratamiento farmacológico del cáncer de ovario epitelial en México / Recommendations on the pharmacological treatment of epithelial ovarian cancer in Mexico

Resumen El cáncer de ovario es la tercera neoplasia maligna ginecológica más frecuente globalmente y también en México, con una elevada tasa de mortalidad debido a que en muchos casos su diagnóstico se realiza en etapas avanzadas. Para establecer su pronóstico es importante la determinación del subtipo y del grado de evolución. En los últimos años, el manejo del cáncer de ovario ha sufrido una importante evolución con la incorporación de nuevas opciones terapéuticas, que a su vez representan un incremento en la supervivencia de estas pacientes. Se presentan las recomendaciones para el manejo del cáncer de ovario elaboradas por un panel de expertos mexicanos basadas en la evidencia disponible hasta el momento y en las características de la atención sanitaria del país.

Abstract Ovarian cancer is the third most frequent gynaecological malignancy worldwide and in Mexico, with a high mortality rate, due to that in many cases its diagnosis is made in advanced stages. Prognosis is important for determining the subtype and the degree of evolution. During lasts years, the management of ovarian cancer has undergone an important evolution with the incorporation of new therapeutic options, which in turn represent an increase in the survival of these patients. We present recommendations for the management of ovarian cancer developed by an expert panel Mexican based on available evidence so far and the characteristics of health care in the country.

Diseño y validación de aspecto y contenido de una escala de riesgo de embarazo no deseado / Design and validation of the look and contents of a scale to measure the risk of unintended pregnancy

OBJETIVO: diseñar y realizar la validación de aspecto y de contenido de un instrumento de valoración del riesgo de embarazo no deseado (END) en mujeres en edad fértil. MÉTODO: estudio descriptivo transversal para la elaboración y validación del aspecto y contenido de una escala en tres fases. Se realizó una revisión bibliográfica para la selección de ítems que permitió elaborar un borrador de la escala, sometido a validación mediante panel de expertos y prueba piloto. Se llevó a cabo un estudio preliminar en el que se administró la versión definitiva de la escala a 60 mujeres en edad fértil. Se realizó estadística descriptiva en las distintas fases. RESULTADOS: la primera versión del cuestionario constaba de 17 ítems. Tras la valoración de los expertos y la prueba piloto se reformularon siete y se añadieron tres nuevos, conformando una segunda versión con 20 ítems. En la prueba piloto destacó como criterio mejor valorado el tiempo empleado para realizar el cuestionario con media de 4,6 sobre 5 (DE 0,47). Se modificaron seis ítems para ganar comprensión. En el estudio preliminar, el intervalo de valores obtenidos en la escala fue de 8-23 puntos. El 81,7% de la muestra se agrupó en los valores 12-18. CONCLUSIONES: se presenta una escala para la estimación del riesgo de END con adecuada validez de aspecto y de contenido, siendo sencilla y viable su administración en mujeres en edad fértil. Es pertinente continuar con el estudio para validar el resto de propiedades psicométricas de la herramienta y confirmar su fiabilidad

OBJECTIVE: to design and conduct validation for the look and contents of a tool to assess the risk of unintended pregnancy (UIP) among women of childbearing age. METHOD: a descriptive cross-sectional study for the preparation and validation of the look and contents of a scale in three stages. A bibliographic review was conducted in order to select items, which allowed to prepare a draft scale, submitted to validation through a board of experts and a pilot test. A preliminary study was conducted, where the final version of the scale was applied to 60 women of childbearing age. Descriptive statistics was conducted at the different stages. RESULTS: the first version of the questionnaire included 17 items. After the assessment by experts and the pilot test, seven were reworded, and three new items were added, leading to a second version with 20 items. In the pilot test, the best valued criterion was the time used to complete the questionnaire, with a mean 4.6 out of 5 (SD 0.47). Six items were modified in order to improve their understanding. In the preliminary study, the range of scores obtained in the scale was from 8 to 23 points; 81.7% of the sample was grouped in the 12 to 18 scores. CONCLUSIONS: a scale was presented for the assessment of the risk of UIP, with adequate validation for design and contents, and easy and feasible to apply in women of childbearing age. It is relevant to continue the study in order to validate the rest of psychometric properties of the tool, and confirm its reliability

Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse.

Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.

Phase 2 Study of Trabectedin in Patients With Hormone Receptor-Positive, HER-2-Negative, Advanced Breast Carcinoma According to Expression of Xeroderma Pigmentosum G Gene.

BACKGROUND: Preclinical and clinical data suggest that xeroderma pigmentosum G gene (XPG) status might predict trabectedin efficacy. This phase 2 study evaluated the efficacy of trabectedin at a dose of 1.3 mg/m2 as a 3-hour intravenous infusion every 3 weeks in hormone receptor-positive, HER-2 (human epidermal growth factor receptor 2)-negative, advanced breast cancer patients according to the tumor level of XPG mRNA expression. PATIENTS AND METHODS: Patients were stratified into high-XPG (>3) or low-XPG (≤ 3) groups. The primary efficacy end point was progression-free survival (PFS) rate at 16 weeks (PFS4); secondary efficacy end points were overall response rate (ORR), duration of response, PFS, overall survival, and safety of trabectedin in this patient population. RESULTS: Forty-four patients were treated, 21 with high XPG and 23 with low XPG. Four high-XPG and 6 low-XPG patients experienced PFS4; the criterion for further recruitment (> 6 patients experienced PFS4) was thus not met, and recruitment was stopped. One high-XPG patient had a partial response (ORR, 5%). One low-XPG patient had a complete response, and 2 low-XPG patients had partial responses (ORR, 13%). Comparison of efficacy parameters between high-XPG and low-XPG patients showed no statistically significant differences. ORR in the efficacy population was 9.3%, median PFS was 1.9 months, and overall survival was 11.8 months. The safety of trabectedin in breast carcinoma was similar to that shown in other indications. CONCLUSION: Trabectedin as single agent had limited activity in hormone-positive, HER-2-negative advanced breast cancer. XPG mRNA expression was not predictive of trabectedin efficacy.

Phase I, dose-escalating study of elisidepsin (Irvalec(®)), a plasma membrane-disrupting marine antitumor agent, in combination with erlotinib in patients with advanced malignant solid tumors.

OBJECTIVE: To determine the recommended dose for phase II trials of elisidepsin (PM02734, Irvalec®) in combination with erlotinib in patients with advanced malignant solid tumors. METHODS: Open-label, dose-escalating, phase I study of intravenous elisidepsin administered weekly (days 1, 8 and 15) over 3 h as a flat dose (FD) and daily oral erlotinib, every 3 weeks. A pharmacokinetic analysis was done on blood samples collected around the first elisidepsin infusion. RESULTS: Thirty patients were treated across six different dose levels (DLs) ranging from elisidepsin 0.33-2.25 mg/erlotinib 100-150 mg. Two patients had dose-limiting toxicities: grade 3 bilirubin increase (DL3: 0.75 mg/150 mg) and a dose omission for > 2 weeks due to grade 3 alanine aminotransferase increase (DL6: 2.25 mg/100 mg). The daily erlotinib dose was escalated to 150 mg at DL2-DL5, but decreased to 100 mg at DL6, as most grade 3 toxicities were related to this agent only. The most frequent toxicities were transaminase increases (related to elisidepsin), and rash, pruritus and diarrhea (related to erlotinib). No objective responses were observed. Despite no overlapping toxicities, the combination was declared unfeasible due to frequent elisidepsin dose delays. The pharmacokinetics of elisidepsin/erlotinib was not significantly different from that of each agent alone. CONCLUSION: The difficulty in combining elisidepsin with the standard dose of erlotinib (150 mg), together with the lack of antitumor activity, made the combination unattractive for further development. The trial was closed without having determined a recommended dose.

Efecto de la vitrificación de ovocitos humanos sobre la capacidad de unión y el estado acrosomal de espermatozoides humanos / Effect of the vitrification of human oocytes on the binding capacity and acrosome status of human sperm

Conocer los aspectos moleculares que acontecen en el proceso de unión de los espermatozoides humanos a la zona pelúcida (ZP) humana es uno de los grandes retos de la biología de la Reproducción. Por otra parte conocer si el proceso de fecundación puede verse afectado por la criopreservación de los gametos femeninos sigue siendo otra cuestión debatida en la literatura. En base a esto, el objetivo principal de este trabajo fue conocer si la vitrificación ovocitaria puede alterar la interacción de los espermatozoides con el glicocáliz de la ZP y demostrar si la ZP de estos ovocitos pierde la capacidad de inducir la reacción acrosómica en los espermatozoides. Según nuestros resultados el método de vitrificación ovocitaria cerrado (S3) no altera la capacidad de unión de los espermatozoides a la zona pelúcida, ni la capacidad de ésta para inducir la reacción acrosómica.

To know the molecular aspects that occur in the process of human sperm binding to the human zona pellucida (ZP) is one of the great challenges of reproduction biology. Moreover knowing if the fertilization process may be affected by cryopreservation of female gametes is still another issue discussed in the literature. Based on this, the main objective of this study was to determine whether the oocyte vitrification may alter the interaction of sperm with the glycocalyx of ZP and show whether these oocytes lost the ability to induce the acrosome reaction in sperm. According to our results the oocyte closed vitrification method (S3) does not alter the ability of the sperm binding to the zona pellucida, and their ability to induce the acrosome reaction.

Phase I study of elisidepsin (Irvalec®) in combination with carboplatin or gemcitabine in patients with advanced malignancies.

OBJECTIVE: To determine the maximum tolerated dose and the recommended dose (RD) for phase II trials of elisidepsin (Irvalec®) in combination with carboplatin or gemcitabine. METHODS: Open-label, dose-escalating, two-arm, uncontrolled, phase I study. Patients received carboplatin on Day (D) 1, followed by elisidepsin on D1 and D8, every 3 weeks, or gemcitabine on D1 and D15, followed by elisidepsin on D1 and D15, every 4 weeks. A pharmacokinetic analysis was done from blood samples collected during the first treatment infusion. RESULTS: Fifteen patients were treated with carboplatin/elisidepsin at doses from 4 AUC/1.0 mg flat dose (FD) to 5 AUC/2.5 mg FD. Two patients had dose-limiting toxicities (DLTs) at 5 AUC/2.0 mg, a dose delay >2 weeks due to grade-2 ALT increase and grade-3 thrombocytopenia, and a D8 infusion omission due to grade-3 ALT increase. The RD was established at 4 AUC/1.0 mg. Toxicity consisted mainly of mild-moderate anorexia, fatigue, and nausea. Twenty-two patients were treated with gemcitabine/elisidepsin at doses from 1,000 mg*m(2)/1.0 mg FD to 1,250 mg*m(2)/7.5 mg FD. Two patients had DLTs at 1,250 mg*m(2)/7.5 mg, both a D15 dose omission due to grade-2 ALT increase. The RD was defined at 1,250 mg*m(2)/5.0 mg. Toxicity consisted mainly of mild-moderate fatigue, pruritus, erythema, and myalgia. No objective response was observed. No relevant pharmacokinetic interaction was detected. CONCLUSION: Infra-optimal doses of elisidepsin and carboplatin and a lack of antitumor activity despite using active drug concentrations in combination with gemcitabine do not warrant further clinical development for these two combinations.

Phase II study of weekly PM00104 (ZALYPSIS(®)) in patients with pretreated advanced/metastatic endometrial or cervical cancer.

This open-label, two-arm, phase II clinical trial evaluated the antitumor activity and safety profile of PM00104 (Zalypsis(®)) administered as a 1-h, weekly, intravenous infusion (days 1, 8 and 15; every 4 weeks) at a dose of 2 mg/m(2) to patients with advanced and/or metastatic endometrial (EC) or cervical cancer (CC) after one previous line of systemic chemotherapy. Twelve patients (median age, 61.5 years) with pretreated EC received a median of 2 treatment cycles (range 1-5) and seven patients (median age, 38 years) with pretreated CC received 2 treatment cycles. None achieved objective tumor response. Median progression-free survival (PFS) was 1.8 months, and median overall survival (OS) was 5.5 months in EC (median follow-up = 20.1 months); median PFS was 1.5 months, and median OS was 5.6 months in CC (median follow-up = 17.1 months). The most common toxicities reported were mild to moderate asthenia, nausea, vomiting and diarrhea. Despite PM00104 showing mostly mild, predictable, manageable and reversible toxicity, protocol criteria for further recruitment were not met in EC, a futility analysis was done and recruitment was stopped; a low patient recruitment rate together with no evidence of activity in CC resulted in early study closure.

Chemical composition, starch digestibility and antioxidant capacity of tortilla made with a blend of quality protein maize and black bean.

Tortilla and beans are the basic components in the diet of people in the urban and rural areas of Mexico. Quality protein maize is suggested for tortilla preparation because it presents an increase in lysine and tryptophan levels. Beans contain important amounts of dietary fiber. The objective of this study was to prepare tortilla with bean and assesses the chemical composition, starch digestibility and antioxidant capacity using a quality protein maize variety. Tortilla with bean had higher protein, ash, dietary fiber and resistant starch content, and lower digestible starch than control tortilla. The hydrolysis rate (60 to 50%) and the predicted glycemic index (88 to 80) of tortilla decreased with the addition of bean in the blend. Extractable polyphenols and proanthocyanidins were higher in the tortilla with bean than control tortilla. This pattern produced higher antioxidant capacity of tortilla with bean (17.6 µmol Trolox eq/g) than control tortilla (7.8 µmol Trolox eq/g). The addition of bean to tortilla modified the starch digestibility and antioxidant characteristics of tortilla, obtaining a product with nutraceutical characteristics.

Daily and circadian melatonin release in vitro by the pineal organ of two nocturnal teleost species: Senegal sole (Solea senegalensis) and tench (Tinca tinca).

This research aimed to investigate melatonin rhythms in the pineal organ of two nocturnal fish species, sole and tench, which show high sensitivity to light. Pineal organs were cultured in vitro under an LD (12 h light:12 h dark) cycle to study the daily rhythmicity of melatonin release. In addition, the in vitro culture was performed under conditions of constant darkness (DD) to study the endogenous control of the rhythm. In the pineal organs cultured under an LD cycle, rhythmic melatonin release was evident in both species, with low values observed during the photophase (15.6+/-7.2 and 22.6+/-2.6 pg/mL for sole and tench, respectively) and high values coinciding with the scotophase (74.0+/-8.2 and 82.1+/-9.1 pg/mL, for sole and tench, respectively). Under LD, the rhythm had a period of 24 h (p<0.001) and presented similar acrophases for both species, located around 9-10 h after lights off (2 and 3 h before the end of the dark phase). When the pineal organs were cultured under DD, the results differed between the species studied. A marked circadian rhythm in melatonin release by the pineal was registered in tench, with lower values during the subjective day, i.e. the period that was previously day (6.2+/-1.6 pg/mL) and higher values during the subjective night, i.e. the period that was previously night (20.4+/-5.5 pg/mL). The rhythm had a mean tau of 24.1 h (p<0.01) and the acrophase was located around 12 h after lights off (the beginning of the subjective day), slightly later than that registered under LD conditions. In contrast, melatonin values in sole remained high during darkness (around 92.0+/-6.9 pg/mL) for four consecutive days, including subjective day periods. In short, these findings revealed that the rhythm of melatonin release in tench is under endogenous control by a circadian oscillator within the pineal organ, while no such pacemaker was evident in sole, which melatonin rhythm appeared to be exclusively light-driven.

Immunolocalization and possible functional role of PSP-I/PSP-II heterodimer in highly extended boar spermatozoa.

PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma which is able to preserve, in vitro, the viability, motility, and mitochondrial activity of highly extended boar spermatozoa for at least 5 hours. However, little is known about the binding pattern of the heterodimer to the sperm plasma membrane and its eventual relation with the maintenance of the sperm functionality. The present study investigated the effect of exposing highly extended boar spermatozoa (1 million/mL) to 1.5 mg/mL of PSP-I/PSP-II for 0.5, 5, and 10 hours at 38 degrees C on sperm characteristics and the changes in PSP-I/PSP-II localization as a result of both the addition of PSP-I/PSP-II to the extender and the incubation time. Exposure of the spermatozoa to PSP-I/PSP-II preserved sperm viability, motility, and mitochondrial activity when compared to nonexposed spermatozoa. This protective effect lasted for 10 hours (P < .05). After immunolabeling of highly extended semen with rabbit monospecific polyclonal antibody against PSP-I/PSP-II, the percentage of immunopositive spermatozoa declines over time from 71% (0.5 hours) to 49% (10 hours). However, more than 80% of spermatozoa remained labeled during the 10-hour incubation period if PSP-I/PSP-II was added. Scanning electron microscopy revealed 4 different binding patterns. The heterodimer was mainly localized to the acrosomal area, being redistributed to the postacrosomal area or lost during in vitro incubation. In conclusion, the protective effect of the heterodimer appears to be related to its adhesion to the acrosomal area, and the loss of this protective effect coincides with a stepwise redistribution of PSP-I/PSP-II during incubation.

Dissecting the protective effect of the seminal plasma spermadhesin PSP-I/PSP-II on boar sperm functionality.

To dissect the protective activity of PSP-I/PSP-II, the effect of the isolated subunits PSP-I and PSP-II and their affinity-purified tryptic peptide and glycan fractions on the viability, mitochondrial activity, and motility of highly diluted boar spermatozoa was investigated. High dilution exerted a negative effect on control spermatozoa. Incubation of spermatozoa with PSP-I/PSP-II or with its PSP-II subunit had a protective effect on sperm functionality, high mitochondrial membrane potential, and sperm motility. These effects were less pronounced when spermatozoa were incubated with the PSP-I subunit. It was noteworthy that motility was abolished by incubation of spermatozoa with isolated PSP-I. Trypsin-degraded PSP-I/PSP-II, PSP-I, and PSP-II reproduced the effects of the native proteins. Incubating spermatozoa with the glycan-depleted tryptic-peptide fraction of PSP-I/PSP-II for 5 hours preserved a higher percentage of viable spermatozoa than when sperm was incubated for the same time with the native heterodimer, trypsin-digested PSP-I/PSP-II, the glycan fraction or without added proteins. However, sperm motility decreased as the concentration of added peptide fraction increased. On the other hand, spermatozoa incubated with the glycan fraction showed lower values than spermatozoa incubated with the peptide fraction. We concluded that the subunits of the PSP-I/PSP-II heterodimeric spermadhesin exert different activities on sperm functions. The finding that the beneficial effect of the native PSP-I/PSP-II on the functionality of highly diluted boar spermatozoa is largely preserved in its isolated PSP-II subunit and does not appear to require the glycan moiety points to a peptide moiety as a potential sperm function-preserving additive of highly diluted boar spermatozoa.

Influence of seminal plasma PSP-I/PSP-II spermadhesin on pig gamete interaction.

The seminal plasma PSP-I/PSP-II spermadhesin is able to preserve, in vitro, the viability of highly extended boar spermatozoa, suggesting it might be used as a suitable ameliorator for the damaging effects of sperm handling, including in vitro fertilization. However, little is known about the ligand capability of PSP-I/PSP-II as regards the zona pellucida (ZP) or its possible role in gamete interaction. The present study evaluated the effect of the presence of PSP-I/PSP-II (1.5 mg/ml) during in vitro oocyte maturation and also during co-incubation of frozen-thawed boar spermatozoa with either immature (IM) or in vitro matured (IVM) oocytes, either enclosed by cumulus cells or denuded. Exposure of the gametes to the heterodimer during in vitro gamete co-incubation showed a significant blocking effect of sperm penetration rates and a decreased number of spermatozoa per oocyte in both IM and IVM denuded oocytes. Such an effect was not present in cumulus-enclosed oocytes, suggesting the effect could be mediated by exposed ZP receptors. In addition, when PSP-I/PSP-II was added to the IVM medium, oocyte maturation rates were significantly reduced. In conclusion, the results suggest that PSP-I/PSP-II, when present in vitro, blocks sperm-ZP binding.

Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase.

In the present study the potential benefit of reactive oxygen species (ROS)-scavenging enzymes superoxide dismutase (SOD) and catalase (CAT) when cryopreserving boar spermatozoa was evaluated. Pooled ejaculate sperm-rich fractions collected from 3 fertile boars were frozen in a split design, after being extended in a conventional freezing extender (control) or the same extender supplemented with SOD (150 or 300 IU/mL, experiment 1), CAT (200 or 400 IU/mL, experiment 2), or SOD + CAT in combination (150 + 200 or 300 + 400 IU/mL, experiment 3). Irrespective of the concentration used, SOD and CAT, alone or in combination, significantly improved postthaw sperm survival, in terms of total sperm motility (assessed with CASA) and viability (assessed with a triple stain; propidium iodide/R123/fluorescein isothiocyanate-labeled peanut agglutinin). Moreover, CAT alone, at a concentration of 400 IU/mL, or in combination with SOD, at concentrations of 200 and 400 UI/mL, improved the ability of frozen-thawed spermatozoa to produce embryos in vitro (zygote cleavage and blastocyst formation as end points). Additional data of ROS generation (luminol- and lucigenin-dependent chemiluminescence) and membrane lipid peroxidation (malondialdehyde [MDA] production) indicated that SOD and CAT reduced postthaw ROS generation by boar spermatozoa, without any influence on MDA production.

Does seminal plasma PSP-I/PSP-II spermadhesin modulate the ability of boar spermatozoa to penetrate homologous oocytes in vitro?

Low concentration (0.15 mg per million of spermatozoa) of seminal plasma-derived PSP-I/PSP-II spermadhesin heterodimer is able to preserve the viability of highly extended boar spermatozoa. Whether spermatozoa also keep their fertilizing capacity is not yet known. The present study evaluated the effect of exposing freshly extended and frozen-thawed boar spermatozoa (10 million/mL) to PSP-I/PSP-II (1.5 mg/mL) for 30 or 120 minutes on sperm characteristics and the outcome of in vitro penetration of immature (IM) and in vitro matured (IVM) homologous oocytes, aiming to identify this spermadhesin as a suitable modulator for sperm-handling protocols. Although exposure to the heterodimer improved sperm viability and motility without increasing the levels of sperm acrosome exocytosis in both freshly extended and frozen-thawed spermatozoa, this pretreatment did not affect sperm penetration rates or sperm numbers per oocyte when pretreated fresh spermatozoa were coincubated with IM or IVM oocytes compared with controls. When cryopreserved spermatozoa were tested, however, on IVM oocytes, already a 30-minute preincubation exposure to PSP-I/PSP-II showed a significant blocking effect on penetration rate (from 90% to 32%, P < .05) and on mean sperm numbers per oocyte (2.9 to 1.6, P < .05). To disclose the nature of this paradox, frozen-thawed spermatozoa were cleansed (by centrifugation in saline bovine serum albumin or through Percoll density gradient separation) and the procedure repeated. Oocyte penetration (but not number of spermatozoa per oocyte) increased (P < .05) when spermatozoa were cleansed with Percoll compared with either washed or unwashed controls (53% vs 13% vs 31%, respectively). In addition, the percentages of polyspermic oocytes remained lower than control (38.5% vs 68.7%, respectively; P < .05). In conclusion, the results confirm that exposure of fresh or frozen-thawed boar spermatozoa to a low dose of seminal PSP-I/PSP-II spermadhesin preserves sperm viability and motility in vitro. Although there was no obvious influence of the heterodimer on the capability of freshly extended boar spermatozoa to penetrate homologous oocytes (either IM or IVM), PSP-I/PSP-II exerted a deleterious effect when frozen-thawed spermatozoa were used to penetrate IVM oocytes. Such an effect of cryopreservation seems to a certain extent reversible, since cleansing of the sperm surface decreased, at least partially, this blocking effect, increasing both penetration and the monospermic rates.

Influence of porcine spermadhesins on the susceptibility of boar spermatozoa to high dilution.

The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300,000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38 degrees C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.