The molecular chaperone HSPA2 plays a key role in regulating the expression of sperm surface receptors that mediate sperm-egg recognition.

A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm-egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm-zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.

Outcomes from the first assisted reproduction program for HIV-serodiscordant couples in Australia.

OBJECTIVE: To describe the clinical outcomes for all HIV-serodiscordant couples attending an assisted reproduction program. DESIGN, SETTING AND PARTICIPANTS: Retrospective review of demographic, clinical and outcome data for all HIV-serodiscordant couples who attended an assisted reproduction program at a tertiary hospital in Melbourne, between its commencement in 2003 and June 2010. MAIN OUTCOME MEASURES: Pregnancies, miscarriages, births, HIV transmission to the HIV-negative partner, semen quality and detection of HIV (HIV RNA and HIV DNA) in semen. RESULTS: As of June 2010, 39 HIV-positive clients had proceeded to assisted reproduction after the initial consultation in the program. There were 162 completed cycles, with 26 pregnancies (clinical pregnancy rate per cycle, 16.2% for HIV-positive men with an HIV-negative partner, and 15.4% for HIV-positive women). Of all 222 tested semen samples, 18 (8%) had HIV RNA detected despite these men receiving antiretroviral therapy and having an undetectable HIV viral load in plasma. Sperm velocity was significantly lower in HIV-positive clients receiving combination antiretroviral therapy than in a control group of recipient-recruited sperm donors (P = 0.01); there were no other significant differences in sperm quality between the two groups. No HIV transmission to babies or HIV-negative partners occurred. CONCLUSION: Our findings show detectable HIV in 8% of semen samples from men with an undetectable HIV viral load in plasma, but confirm the safety of assisted reproduction for HIV-serodiscordant couples within a program with strict protocols for HIV treatment and testing of all semen before use.

Long-term health, well-being, life satisfaction, and attitudes toward parenthood in men diagnosed as infertile: challenges to gender stereotypes and implications for practice.

OBJECTIVE: To investigate attitudes toward parenthood, long-term life satisfaction, and health and well-being in men diagnosed as infertile. DESIGN: A cross-sectional survey of a cohort of men 5 years after diagnosis of infertility. SETTING: The andrology clinic at the Royal Women's Hospital Reproductive Services, Melbourne Australia. PATIENT(S): All men diagnosed at this center as infertile in 2001 and 2002. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Attitudes to parenthood (Meaning of Parenthood), quality of intimate relationship (Intimate Bonds Measure), personality characteristics (Vulnerable Personality Style Questionnaire), life satisfaction (Satisfaction with Life Scale), and self-rated physical health (Physical Component Summary of SF-12 [PCS-12]) and relationship with mental health (Mental Component Summary of SF12 [MCS-12]). RESULT(S): A total of 112 (41%) of 276 men completed the survey. Of these, 96% had pursued infertility treatment and 87% had become fathers. Only 10% thought that fertility confirmed by fatherhood reflected masculinity, and 84% desired parenthood as much as their partners did. When all other factors were controlled for, men who had not become fathers had poorer mental health (MCS-12 score = 43.9 +/- 9.9) than those who were fathers (MCS-12 score = 49.25 +/- 8.7). CONCLUSION(S): Clinical practice should not presume that infertile men conflate fertility and masculinity, are less distressed than women about the potential loss of parenthood, or adjust more readily to childlessness, which appear to be inaccurate but widespread stereotypes.

Preterm birth, ovarian endometriomata, and assisted reproduction technologies.

OBJECTIVE: To report preterm birth and small for gestational age (SGA) rates from assisted reproduction technologies (ART) patients with ovarian endometriomata compared with control groups. DESIGN: Retrospective cohort study. SETTING: Tertiary university affiliated ART center and Perinatal Data Collection Unit (PDCU). PATIENT(S): Every woman who had an ART singleton baby born between 1991 and 2004 had her database record assessed (N = 4382). Control groups included 1201 singleton babies from ART patients without endometriosis and 2400 randomly selected women from the PDCU database of 850,000 births. INTERVENTION(S): There were 95 singleton ART babies from patients with ovarian endometriomata and 535 ART singleton babies from patients who had endometriosis but no ovarian endometriomata. MAIN OUTCOME MEASURE(S): Preterm birth rates and SGA birth rates. RESULT(S): Preterm birth rate increased only in the ovarian endometriomata group when compared with community birth records (n = 850,000). Furthermore, ART patients with ovarian endometriomata had a statistically significantly increased likelihood of having a SGA baby when compared with other forms of endometriosis. CONCLUSION(S): Rates of preterm birth and SGA babies doubled in infertility patients with ovarian endometriomata who required ART.

Antenatal mood and fetal attachment after assisted conception.

OBJECTIVE: Australian women conceiving with ART are at fourfold risk of admission to early parenting treatment programs compared with those conceiving spontaneously. This study aimed to identify prevalence and determinants of antenatal mood disturbance and other risks for early parenting difficulties after assisted conception. DESIGN: A prospective longitudinal investigation from conception to 18 months postpartum using telephone interviews and self-report questionnaires. SETTING: Melbourne IVF and Royal Women's Hospital Reproductive Services, Victoria, Australia. PATIENT(S): A consecutive cohort of English-speaking women with ultrasound-confirmed ART-conceived pregnancies. MAIN OUTCOME MEASURE(S): Standardized psychometric measures of mood, quality of marital relationship, mother-to-fetus emotional attachment, and personality. INTERVENTION(S): None. RESULT(S): Of the 288 women with confirmed pregnancies, 239 were contactable, and 183 (77%) were recruited, 95% of whom completed both early and late pregnancy assessments. Participants were socioeconomically advantaged, had very good pregnancy health, exceptional marital relationships, normal personality styles, and intense affectionate attachment to the fetus. Very few (<5%) had clinically significant mood disturbance in late pregnancy. CONCLUSION(S): There were low rates of antenatal mood disturbance and other risk factors for postpartum depression. Pregnancy and motherhood might be idealized after ART conception, and preparation for the realities of infant care might then be insufficient.

In vitro study of heat production during power reduction of equine mandibular teeth.

OBJECTIVE: To measure the amount of heat generated during 3 methods of equine dental reduction with power instruments. DESIGN: In vitro study. SAMPLE POPULATION: 30 premolar and molar teeth removed from mandibles of 8 equine heads collected at an abbatoir. PROCEDURE: 38-gauge copper-constantan thermocouples were inserted into the lingual side of each tooth 15 mm (proximal) and 25 mm (distal) from the occlusal surface, at a depth of 5 mm, which placed the tip close to the pulp chamber. Group-NC1 (n = 10) teeth were ground for 1 minute without coolant, group-NC2 (10) teeth were ground for 2 minutes without coolant, and group-C2 (10) teeth were ground for 2 minutes with water for coolant. RESULTS: Mean temperature increase was 1.2 degrees C at the distal thermocouple and 6.6 degrees C at the proximal thermocouple for group-NC1 teeth, 4.1 degrees C at the distal thermocouple and 24.3 degrees C at the proximal thermocouple for group-NC2 teeth, and 0.8 degrees C at the distal thermocouple and -0.1 degrees C at the proximal thermocouple for group-C2 teeth. CONCLUSIONS AND CLINICAL RELEVANCE: In general, an increase of 5 degrees C in human teeth is considered the maximum increase before there is permanent damage to tooth pulp. In group-NC2 teeth, temperature increased above this limit by several degrees, whereas in group-C2 teeth, there was little or no temperature increase. Our results suggest that major reduction of equine teeth by use of power instruments causes thermal changes that may cause irreversible pulp damage unless water cooling is used.

Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line.

AIM: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. METHODS: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed. RESULTS: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZP1 was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZP1 was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed. CONCLUSION: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZP1 from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.

Induction of spermatogenesis by recombinant follicle-stimulating hormone (puregon) in hypogonadotropic azoospermic men who failed to respond to human chorionic gonadotropin alone.

A multicenter, open-label, randomized efficacy and safety study was performed with combined human chorionic gonadotropin (hCG) and recombinant follicle-stimulating hormone (recFSH) (Puregon(R)) treatment to induce spermatogenesis in hypogonadotropic hypogonadal male patients. Patients were pretreated for 16 weeks with hCG to normalize testosterone levels. A total of 30 of 49 (61%) subjects had normalized testosterone levels but were still azoospermic after the hCG-alone phase. These patients were randomized into 2 treatment schemes with recFSH (2 x 225 IU recFSH per week [group A] or 3 x 150 IU recFSH per week [group B]), in combination with hCG for a period of 48 weeks. Total testosterone increased during the hCG-alone period from 1.08 and 1.22 ng/mL to 6.26 and 4.52 ng/mL for groups A and B, respectively. Combined gonadotropin treatment was effective in inducing spermatogenesis (sperm count >/=1 x 10(6)/mL) in 14 of 30 subjects (47%) and this was achieved after a median duration of treatment of approximately 5.5 months. Treatment time necessary for first sperm cells to appear in the ejaculate was related to the initial testicular volume. Subjects with a history of maldescended testes (11 of 30 subjects, 37%) showed a lower mean response to treatment as indicated by the relatively lower number of subjects reaching levels of at least 1 x 10(6) sperm cells per milliliter. Combined testicular volume increased during combined gonadotropin treatment from 11.4 to 24.0 mL. Although subjects with a history of maldescended testes had a lower starting testicular volume, subjects with and without a history of maldescended testes showed approximately the same relative increase in testicular volume. Total testosterone levels showed only a minor further increase during the combined gonadotropin treatment period. In conclusion, a weekly dose of 450 IU (3 x 150 IU or 2 x 225 IU) recFSH, in addition to hCG, was able to induce spermatogenesis in many hypogonadotropic azoospermic men who failed to respond to treatment with hCG alone.

In vitro anion transport alterations and apoptosis induced by phenylbutazone in the right dorsal colon of ponies.

OBJECTIVES: To study the functional and structural responses of the right dorsal colon (RDC) of ponies to phenylbutazone (PBZ) in vitro at a concentration that could be achieved in vivo. ANIMALS: 8 adult ponies. PROCEDURE: Short circuit current and conductance were measured in mucosa from the RDC. Tissues incubated with and without HCO3- were exposed to PBZ, bumetanide, or indomethacin. Bidirectional Cl- fluxes were determined. After a baseline flux period, prostaglandin E2 (PGE2) was added to the serosal surfaces and a second flux period followed. Light and transmission electron microscopy were performed. RESULTS: Baseline short circuit current was diminished significantly by PBZ and indomethacin, and increased significantly after addictions of PGE2. After PGE2 was added, Cl- secretion increased significantly in tissues in HCO3- -free solutions and solutions with anti-inflammatory drugs, compared with corresponding baseline measurements and with control tissues exposed to PGE2. Bumetanide did not affect baseline short circuit current and Cl- fluxes. The predominant histologic change was apoptosis of surface epithelial cells treated with PBZ and to a lesser extent in those treated with indomethacin. CONCLUSIONS AND CLINICAL RELEVANCE: Prostaglandin-induced Cl- secretion appeared to involve a transporter that might also secrete HCO3-. Both PBZ and indomethacin altered ion transport in RDC and caused apoptosis; PBZ can damage mucosa through a mechanism that could be important in vivo. The clinically harmful effect of PBZ on equine RDC in vivo could be mediated through its effects on Cl- and HCO3- secretion.

Comparison of two heated gutta-percha obturation techniques of the mandibular first molar in the dog.

An in vitro study compared two heated gutta-percha obturation techniques of the mandibular first molar in the dog. Thirty hemimandibles were harvested from adult dog cadavers. The mandibular first molars were instrumented and obturated using either the ThermaFil Plus technique or the SuccessFil vertical compaction technique. Obturation times were recorded and three radiographic views were taken of all teeth for evaluation of the endodontic fill. Fifty-two of the mandibular first molar roots were evaluated in the apical dye leakage portion of the study. The ThermaFil Plus obturation technique required less time and provided a better radiographic endodontic fill, however there was significantly greater apical dye leakage in this technique compared with teeth that were obturated using the SuccessFil vertical compaction technique.