A follow-up expanded study of the correlation of sperm velocity in seminal plasma and offspring gender.

A preliminary study reported finding higher sperm velocity in seminal plasma in males of partners that conceived female offsprings. The null hypothesis was that sperm velocity was not related to the offspring gender. The objectives were: (a) to expand the previous study, and (b) to correlate offspring gender results with motility parameters determined through the computer-aided sperm analyzer (CASA) system. In combined fresh and frozen cycles (N = 187), sperm from cases with all female offsprings displayed higher curvilinear (48 +/- 1.0 mu/sec versus male 46 +/- 1.0, P < 0.05) and average path velocities (36 +/- 0.7 mu/sec versus male 34 +/- 0.7, P < 0.01). A criteria of less than 30 mu/sec or over 41 mu/sec average path velocity predicted 73 or 72% of the male or female offspring cases, respectively. A curvilinear velocity of less than 49 mu/sec or over 55 mu/sec predicted 58 or 59 % of the male or female offspring cases, respectively. Semen viscosity reflected in sperm velocity was linked to predominantly male or female sperm populations. Paracrine signals from the gender-skewed sperm precursor populations controlling viscosity merit further exploration.

Development of mammalian embryos exposed to mixed-size nanoparticles.

Inhaled or ingested ultrafine nanoparticles and their effects on early pregnancy remain polemic. The objectives of the study were: (a) to determine the embryotoxic effects of nanoparticles at the 2-cell stage and (b) to localize the internalized nanoparticles in the blastocyst. Thawed mouse 2-cell embryos (no. = 128) were exposed to either mixed-size polystyrene-based nanoparticles (11 million/ml) or control G1.3 medium and assessed after 72 hours. Additionally, blastocysts (no. = 146) were exposed to nanoparticles and analyzed. The results showed that the nanoparticles did not inhibit 2-cell embryo development to the blastocyst stage (89.4 vs 96.8%; treated vs control). There were no differences in hatching (34.8 vs 43.5%), implantation (13.6 vs 24.2%) and degeneration (10.6 vs 3.2%). Delayed exposure to nanoparticles showed similar percent hatching (40.7 vs 47.3%) and implantation (17.6 vs 20.0%). Although nanoparticles were internalized, embryo development was not inhibited suggesting a lack of embryotoxicity. During hatching, the larger nanoparticles adhered to the extruding blastocyst, preferentially on trophoblasts, but interference was insignificant. Exposure to polystyrene-based nanoparticles at the concentration tested are not associated with embryonic loss.

Correlation of sperm parameters with apoptosis assessed by dual fluorescence DNA integrity assay.

Failed fertilization after intracytoplasmic sperm injection or miscarriages occurs in cases involving apoptotic and necrotic sperm. Identifying normal sperm is important for successful assisted reproductive technologies (ART) procedures. The study was conducted to correlate sperm parameters with intact sperm with normal DNA assessed by the dual stain assay in 118 separate individuals. The results showed differences in percent DNA intact sperm in individuals with normal W.H.O. sperm features (62 +/- 1.1; mean +/- S.E.M.) compared with oligoasthenoteratozoospermia patients (38 +/- 5.3). Individuals whose sperm had fertilizing capacity had higher percentages of intact DNA (60 +/- 1.3 versus 47 +/- 2.4). The percentages of intact DNA sperm were significantly correlated to total motility in semen (R = 0.7), post-wash motility (R = 0.6), rapid progression (R = 0.6), intact acrosome (R = 0.5), and strict morphology (R = 0.5). There were no correlations with the remaining parameters. The dual stain assay identified sperm with normal physiology and fertilizing capacity. The dual stain assay measures DNA integrity and is a promising method to select normal sperm for ART.

Cardiovascular effects of topical ophthalmic 10% phenylephrine in dogs.

OBJECTIVE: To evaluate the effect of topical ophthalmic 10% phenylephrine on systolic arterial pressure (SAP), diastolic arterial pressure (DAP), mean arterial pressure (MAP), pulse rate (PR) and electrocardiogram (ECG) in dogs. ANIMALS STUDIED: Nine clinically normal dogs. PROCEDURE: Arterial catheters were placed in the dorsal pedal artery of awake dogs and ECG leads were attached. After a 15-min acclimatization period, baseline PR, SAP, DAP and MAP were recorded every 5 min for 20 min. Two treatment groups (eight dogs each) were studied. Group I: one drop of phenylephrine was placed in each eye once. Group II: one drop of phenylephrine was placed in each eye three times at 5-min intervals. Following treatment, PR, SAP, DAP and MAP were recorded every 5 min for 90 min. The mixed procedure of the SAS system was used to perform a repeated measures analysis of variance to test for linear and quadratic trends across time. RESULTS: Group I: There was a significant quadratic decrease in PR across time (P = 0.0051). Systolic arterial pressure increased linearly with time (P = 0.0002), MAP increased linearly with time (P = 0.0131), and DAP increased linearly with time (P = 0.0001). Group II: There was a significant quadratic decrease in PR across time (P = 0.0023). There was a significant quadratic increase in SAP (P = 0.0324), MAP (P = 0.0103) and DAP (P = 0.0131) across time. CONCLUSIONS: Topical ophthalmic application of 10% phenylephrine in normal dogs results in elevation of arterial blood pressure and reflex bradycardia.

Sperm apoptosis in nonpregnant luteal phase sera after in vitro fertilization as assessed by comparative genomic hybridization.

Toxicity in serum has been reported in cases of recurrent spontaneous abortions and endometriosis. The null hypothesis was that serum toxicity was not involved in failed pregnancies after in vitro fertilization procedures. The objective was to expose donor sperm to pregnant versus nonpregnant patient sera and analyze for sperm DNA damaging effects using a novel comparative genomic hybridization method. Luteal phase sera (N = 21 cases) were drawn one week after embryo transfer. Colloid-washed donor sperm were incubated (48 h, 37 degrees C, 5% CO2 in air) in 0% or 50% sera. Single-stranded DNA (ssDNA) of control sperm were stained in Hoechst 33342 and hybridized to Sybr Gold-stained ssDNA of sera-treated sperm. Image analyses were performed and fluorescent intensities analyzed. Nonpregnant patient sera (57% of cases) were associated with DNA fragmentation (64.4 +/- 8.8 pixels; mean +/- S.E.M.) when compared with pregnant patient sera (106.3 +/- 8.4 pixels). There were no differences in the sera of biochemical (108.2 +/- 15.3) versus clinical pregnancy cases (105.3 +/- 11.4). The results suggest that nonpregnant patient sera contained factor(s) that cause DNA fragmentation leading to pregnancy losses.

Sperm velocity in seminal plasma and the association with gender of offspring.

The gender of the offspring is determined by the fertilizing sperm. Previous gender studies were based on washed sperm, but not on sperm in seminal plasma. The objective was to correlate motility parameters assessed during semen analyses with the offspring gender. For comparison, fixed sperm head DNA quantitated by Hoechst 33342 fluorescence microscopy was also analyzed. Forty-six patients undergoing assisted reproduction procedures resulted in livebirth deliveries with either male or female-predominant offsprings. Sperm head fluorescence was weakly correlated to the gender in 61% of the cases. Sperm of patients with male offsprings had slower curvilinear (44.2 +/- 1.8 mean +/- SEM, versus, 49.9 +/- 2.7 micro /sec) and slower average path velocities (32.4 +/- 1.2 versus 36.3 +/- 1.7 micro /sec). Using cut-off values for the curvilinear (< 49 micro /sec) and average path (< 36 micro /sec) velocities of sperm swimming in seminal plasma, the two parameters predicted 75 and 68% of the male offspring births, respectively. The data suggest that sperm movement in seminal plasma is a marker for factors that skew the ratio of the X- to Y-sperm populations.

Cryopreservation of human cumulus cells for co-cultures and assessment of DNA damage after thawing using the comet assay.

PURPOSE: Cumulus cells have been shown to be beneficial for blastocysts formation in co-cultures but information on cumulus cryopreservation is lacking. The objective was to use the fixed cell comet assay to analyze for DNA damage in cumulus cells after cryopreservation. METHODS: Discarded cumulus cells from follicular aspirates obtained during assisted reproduction procedures (N = 4 cases) were pooled and cryopreserved in either 40% ethylene glycol and 0.5 M sucrose, 12:20% glycerol-egg yolk medium, 28% glycerol hypoosmolar medium or control medium. The cells were processed and stored in liquid nitrogen for 48 h. The thawed cells were smeared on glass slides, fixed, stained with acridine orange, embedded in a mini-agarose layer, and electrophoresis carried out. Fluorescent images were analyzed. RESULTS: The cumulus tail moment, a calculated index of DNA damage, was significantly lower for each of the three cryoprotectant when compared with the control. The two cryoprotectants containing glycerol were associated with higher cumulus cell viability. However, the glycerol-egg yolk combination yielded the highest cell viability. CONCLUSIONS: The cumulus comet assay demonstrated similar DNA integrity in cells frozen in each of the three cryoprotectants. The glycerol-egg yolk medium had the highest cell viability with little or no DNA damage after freeze-thaw. More studies are needed to examine the long-term effect of the cryoprotectants on thawed cumulus cell viability.

Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection.

This paper postulates that in the ovary, the close association between the cumulus cells and the oocytes permits the fertilizing ability of the oocytes to be determined indirectly through cumulus cell DNA status. The objective was to use a modified comet assay to analyse cumulus cell DNA and relate the data to oocyte fertilization after intracytoplasmic sperm injection (ICSI) procedures. Oocytes were retrieved (n = 15 cases) and free-floating cumulus cells were pooled and smeared on clear glass slides to dry. Meanwhile, the denuded oocytes were injected with spermatozoa and fertilization was assessed, followed by embryo transfer. The fixed cumulus cells were stained in acridine orange, coated with a mini-gel agarose layer, lysed in alkaline buffer and electrophoresis performed. Analyses of fluorescent cell images (n = 449) showed that the tail moment was positively correlated to the percentage of fertilization after ICSI (r = 0.567, P < 0.05). In contrast, there was no correlation between tail moment and number of oocytes retrieved, total ampoules used, endometrial thickness and age of patient. The results suggested that the competence of the oocytes was associated with the cumulus cell DNA status. A unique feature here was the comet assay for archived material with obvious advantages.

A comparison of heat-induced hyperactivation in patients' sperm after colloid or pentoxifylline wash methods.

OBJECTIVE: Our purpose was to compare kinematic parameters of human sperm after processing through two different wash methods and 40 degrees C heat treatment. STUDY DESIGN: Sperm specimens (N = 169 cases) were washed by either colloid or pentoxifylline wash methods, and the motility parameters were measured at either 37 degrees C or 40 degrees C at baseline (0 hours) and after 4 hours. Five randomly selected washed specimens with matching 37 degrees C (control) or 40 degrees C heat treatments were assessed for changes in a sentinel gene. RESULTS: The percentage of sperm hyperactive motility was >5 times higher after the 40 degrees C heat treatment, in comparison with the 37 degrees C treatment, for both the colloid- and the pentoxifylline-washed sperm. The percentages of total motility and progression were equally enhanced in heated sperm for the two wash methods. No changes were detected in the sentinel gene with the heat treatment. CONCLUSION: Sperm cells mildly heated at 40 degrees C responded with greater motility, progression, and hyperactivation. The data suggest that mild heat is a stimulus for sperm function because greater sperm hyperactivation is associated with increased sperm fertilizing capacity. The absence of change in the sentinel gene in heated sperm suggests that a temperature of 40 degrees C is too low to initiate alterations in the highly condensed sperm chromatin. More studies are needed before mild heating of ejaculated sperm becomes acceptable for use in assisted reproductive technologies.

A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes.

OBJECTIVE: To correlate sperm variables with sperm DNA fragmentation, as assessed by using a modified alkaline comet assay for sperm smears. DESIGN: The comet assay was adapted for fixed sperm smears (59 cases), and the level of DNA fragmentation was determined. SETTING: Clinical and academic research environment. PATIENT(S): 59 patients undergoing fertility treatment. INTERVENTION(S): Sperm samples leftover from IVF procedures were fixed and processed for the comet assay. MAIN OUTCOME MEASURE(S): Sperm head DNA density and sperm variables. RESULT(S): A correlation was observed between increased sperm head DNA fragmentation and decreased penetration of zona-free hamster oocytes. Heat-induced hyperactive motility decreased as DNA fragmentation increased. The DNA fragmentation did not correlate with percentages of intact acrosome, normality, maturity, and strict normal morphology. CONCLUSION(S): The advantages of the comet assay for archived cells include simplicity, low intraassay coefficient of variation, and low performance cost; in addition, DNA analysis can be carried out at leisure. Low DNA damage was associated with higher hyperactivation and oocyte penetration, suggesting that failed fertilization was linked to compromised DNA integrity in the sperm. Exploration of compounds to repair damaged DNA is warranted.

Roles of prolactin and gonadotropin-releasing hormone in rheumatic diseases.

PRL is capable of influencing immune responses and is a cytokine in all likelihood. Circulating PRL is elevated in a number of autoimmune diseases, and about 20% of SLE patients are hyperprolactinemic. The serum PRL concentration often does not reflect disease activity in SLE. The PRL-suppressing drug bromocriptine has been reported to benefit small numbers of patients with reactive arthritis and inflammatory eye disease, and bromocriptine may be beneficial in treating SLE. In NZB/NZW mice, bromocriptine was beneficial and prolonged life. Bromocriptine therapy favorably modified disease in human SLE. In a preliminary open-label study, SLE patients treated with bromocriptine for 6 months had significant improvement in disease activity. These responses were corroborated by masted therapeutic studies. Daily treatment with low-dose bromocriptine prevented lupus flares, and bromocriptine was as effective as hydroxychloroquine in treating active nonorgan-threatening disease. The reports of the efficacy of bromocriptine treatment of SLE are encouraging. Additional studies may confirm the findings reported in this review and may lead to further use of hormonal modification to treat lupus and other autoimmune diseases. For the present, it is important to understand that treatment with dopamine agonists such as bromocriptine is experimental and best confined to therapeutic trials. In the experience of the authors, bromocriptine should not be relied on to treat severe life-threatening autoimmune disease. If bromocriptine is used to treat SLE and is then discontinued, the patient should be observed carefully for rebound hyperprolactinemia and the development of a lupus flare. GnRH is produced by lymphocytes and exerts immunomodulatory actions. Thus, GnRH resembles a cytokine. GnRH can be shown to exert gender-restricted immune actions in vitro and in vivo. The authors' preliminary observations are consistent with the possibility that gender-related differences in expression of the GnRH receptor or in GnRH signal transducers may contribute to gender-related differences in immune responsiveness to GnRH. These differences in G proteins may contribute to the gender-related differences in immunity and expression of autoimmune disease.

Growth during and after a trial of growth hormone releasing hormone 1-29 in children with idiopathic short stature or growth hormone neurosecretory dysfunction.

The growth promoting effects of once nightly subcutaneous injections of growth hormone releasing hormone (GHRH) 1-29 (30 microg/kg) for 6 months were studied in 16 slowly growing prepubertal children with idiopathic short stature (ISS; Group 1) and 8 similar children with growth hormone neurosecretory dysfunction (GHND; Group 2). Each child underwent endogenous growth hormone evaluation using both pharmacological and physiological testing; each had stimulated values > 10 microg/l and were subsequently placed into one of two groups based on pooled 12-hour overnight GH of < or > or = 3 microg/l. Each patient was followed every three months for one year. There were no significant differences in the two groups throughout the study with the exception of the endogenous GH levels. Both groups responded to GHRH therapy with similar significant increases in their rates of growth. Although a subset of patients (6 of 21) continued to grow at a rate significantly greater than the pre-therapy rate of growth, overall rates of growth were not significantly different from the pre-therapy growth rates 6 months following the discontinuation of GHRH treatment. We conclude that GHRH 1-29, given in the doses provided, leads to similar changes in growth rates in short, slowly growing children who are GH sufficient and those with GHND. Despite prior reports to the contrary, GHND patients do not experience a sustained increased in growth rate upon discontinuation of GHRH.

Analysis of papillomavirus consensus L1 gene in a closed colony of baboons (Papio anubis).

OBJECTIVE: The purpose of this study was to analyze cervical specimens and semen from a closed colony of baboons for the presence of the papillomavirus consensus L1 gene. STUDY DESIGN: Cervical swabs were collected from lightly anesthetized female baboons. Semen was collected from a male baboon by standard electroejaculation techniques. Deoxyribonucleic acid was extracted from the cells by two different methods and analyzed by polymerase chain reaction targeting the L1 consensus gene common for >25 genital papillomaviruses. RESULTS: Analyses of the polymerase chain reaction-amplified products did not reveal bands for the papillomavirus in either the cervical specimens or the semen. CONCLUSIONS: The hypothesis of a linkage between primates with papillomavirus as a common factor is not supported by the results of this study. This information is also important in assisting clinicians in setting up specific pathogen-free colonies of baboons.

Gonadotropin-releasing hormone and G proteins: potential roles in autoimmunity.

The hypothalamic homone gonadotropin-releasing hormone (GnRH) displays gender-specific actions. Pituitary responsiveness to GnRH is generally increased by estrogens and decreased by androgens. GnRH is now known to be produced by the immune system and to exert potent immunologic actions. Our central hypothesis is that gender differences in responsiveness to GnRH in the immune system play a pivotal role in the gender differences in immunity and autoimmunity. Studies in lupus-prone mice demonstrate that GnRH exacerbates murine lupus in a gender-specific fashion. Subsequent studies from our laboratory suggest that the gender differences in immunologic responsiveness to GnRH may relate to differences in the expression of the signal transducers through which GnRH acts, namely, the G proteins, Gs, and Gq/11. We have further demonstrated gender differences in second messengers for GnRH, IP3, and cAMF in immune cells. We have also demonstrated that GnRH agonist increases the quantities and/or activity of G proteins in immune cells in a gender-specific fashion. We speculate that gender differences in GnRH production and action, and in G protein expression play a role in a variety of autoimmune diseases that affect females predominantly.

Gonadotropin-releasing hormone increases CD4 T-lymphocyte numbers in an animal model of immunodeficiency.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) possesses immunostimulatory properties. We have previously demonstrated that GnRH antagonists decrease lymphocyte numbers in an animal model of autoimmune disease. We speculated that the converse might be true, that GnRH administration would increase lymphocyte numbers or alter lymphocyte subsets in an immunodeficiency state. OBJECTIVE: Our purpose was to test the hypothesis that GnRH agonist would increase IgG and CD4 counts in a rat model of immunodeficiency independently of gonadal steroids. METHODS: We used diabetes-prone (DP) BB rats. This model has been characterized to have an AIDS-like lymphocyte profile, with lymphopenia and depressed CD4 counts. Ovariectomized female DP rats were randomized to receive subcutaneous injections with GnRH or vehicle 6 times weekly. DR rats were ovariectomized and treated with vehicle as controls. We performed flow cytometric analysis and complete blood cell counts at baseline, 3.5 weeks, and 7 weeks of treatment. We also measured total serum IgG and luteinizing hormone levels. RESULTS: GnRH administration significantly increased total serum IgG levels in DP rats compared with vehicle. The percentages of CD4(+) cells in blood were also significantly increased in the GnRH-treated group compared with the vehicle-treated group and compared with baseline. Similarly, the absolute numbers of CD4(+) positive T cells were increased over controls at 7 weeks. The effects of GnRH were specific for the CD4 subset because there were no significant differences in numbers of CD8(+) positive cells between the 2 treatment groups. CONCLUSION: GnRH shows potential utility as an immunostimulatory agent in immunodeficient states manifesting diminished numbers of immunocompetent CD4(+) T lymphocytes.

Gender-specific exacerbation of murine lupus by gonadotropin-releasing hormone: potential role of G alpha(q/11).

We have previously demonstrated that GnRH and its analogues modulate the severity of murine systemic lupus erythematosus. In the present study, we demonstrate that GnRH alters disease severity in a sexually dimorphic fashion, even in gonadectomized mice. GnRH administration leads to an exacerbation of lupus in ovariectomized females, whereas it exerts no effect in castrated males. We initially hypothesized that gender differences in lymphocytic expression of GnRH receptor might explain these observations. Using competitive RT-PCR and binding studies to quantitate GnRH receptor expression in lymphoid organs, we found that GnRH administration led to decreased expression of GnRH receptor messenger RNA (mRNA) and GnRH binding, compared with vehicle, in spleens of ovariectomized females after 2 weeks of treatment. These decreases occurred concurrently with increased expression of interleukin-2 receptor mRNA and protein in females. GnRH administration did not alter GnRH receptor or interleukin-2 receptor mRNA or protein in castrated males. GnRH exerts actions on the pituitary through G protein signal transduction, specifically through G alpha(q/11). Competitive RT-PCR revealed that GnRH administration was associated with increases in the expression of G alpha(q/11) mRNA, compared with vehicle, in spleens in ovariectomized females but not in castrated males. Immunoblot analysis revealed a similar pattern. We conclude that gender differences in expression of G alpha(q/11) may contribute to gender differences in immunity and/or autoimmune disease.

Spermac stain analysis of human sperm acrosomes.

OBJECTIVE: To study the association between low percentages of intact sperm acrosomes and fertilization failures in conventional IVF procedures. DESIGN: A retrospective study. SETTING: Clinical and academic research environment. PATIENT(S): Patients undergoing treatment of infertility. INTERVENTION(S): Sperm cells were fixed and stained using the Spermac stain. MAIN OUTCOME MEASURE(S): Percentages of intact acrosomes and fertilization. RESULT(S): There was a significant association between specimens with <40% intact acrosomes and failed conventional IVF procedures. Among the 29 cases with <40% intact acrosomes, 9 cases (31%) resulted in zero penetration of the oocytes. The mean (+/-SEM) percentage of fertilization was lower in the abnormal acrosome group (43.3% +/- 6.5%) than in the normal acrosome group (64.1% +/- 5.6%). The status of the sperm acrosome was not correlated with the results of fertilization in intracytoplasmic sperm injection procedures. CONCLUSION(S): Sperm with low percentages of intact acrosomes were associated with failed fertilization. The Spermac stain was useful for assessing acrosomes and identifying possible male factor infertility problems. The results suggested that a minimum percentage of sperm with intact acrosomes are needed for fertilization to occur in vitro.

Detection of pro-opiomelanocortin mRNA in human and rat caudal medulla by RT-PCR.

Pro-opiomelanocortin (POMC) mRNA has been localized in the NTS of the rat, but not in the human or other species. Here, we report that RT-PCR amplification of human caudal medulla RNA generated a distinct band on agarose gels corresponding in size and sequence to the predicted 742-bp POMC PCR product. The 742-bp signal was undetectable following amplification of cortex, amygdala or caudate nucleus RNA. An homologous, 678-bp band was amplified from rat caudal medulla and, unexpectedly, from other brain regions. Competitive RT-PCR demonstrated that POMC cDNA from rat cortex, striatum and cerebellum was 17%, 22% and 45% of caudal medulla levels. These data indicate that the POMC gene is expressed in human caudal medulla and suggest that small amounts of POMC mRNA are present in regions other than the hypothalamus and NTS of rat brain.

Heat-induced hyperactivation.

PURPOSE: The objectives of this study were (1) to determine the sperm hyperactivation and related kinematic parameters at 40 degrees C after using four sperm wash procedures and (2) to correlate the heat-induced hyperactivation data with cases of clinical pregnancies from either artificial insemination or standard in vitro fertilization (IVF). METHODS: Semen samples (n = 51) were collected by ejaculation, and semen analyses were carried out to determine the pretreatment data. Sperm kinematic measurements were performed using the Hamilton Thorn HTM-C computer-aided sperm analyzer. Hyperactivation was determined using the sort module on the HTM-C. Membrane integrity was assessed using the hypoosmotic sperm swelling procedure. Sperm morphology and acrosomal status were also determined using the Spermac stain. Each semen specimen was divided and processed through either the swim-up wash, the 1-h test-yolk buffer (TYB) wash, the 1 mg/ml pentoxifylline stimulant procedure, or the two-layer 90:47% gradient colloidal solution procedure. The washed sperm were incubated at 25 or at 40 degrees C for 4 hr. After incubation, kinematic parameters were assessed for the posttreatment data. Semen specimens were obtained on different occasions for artificial insemination or standard IVF. Data from intracytoplasmic sperm injection cases were not included to avoid confounding factors. Live births and/or pregnancies with fetal heart-beat examined by ultrasound were considered clinical pregnancies. RESULTS: Heat-induced hyperactive motility was significantly higher in sperm of the male partner of pregnant (n = 7) patients compared with nonpregnant (n = 44) patients (mean +/- SE, 10.0 +/- 3.3 versus 5.5 +/- 0.8%) after TYB processing followed by 4 hr of incubation at 40 degrees C. This was also observed after colloid (Percoll) processing (11.6 +/- 4.6 versus 5.8 +/- 0.8%). There were no differences in hyperactivation after 4 hr at 23 degrees C between pregnant and nonpregnant cases. Parameters such as count, volume, motility, viability, and acrosomal status were not different for the groups. However, the percentage of sperm with normal morphology (WHO classification) was twice as high in the pregnant group versus the nonpregnant group. CONCLUSIONS: Heat-induced hyperactivation was associated with fertile sperm and was predictive of pregnancy obtained after artificial insemination or IVF. The association was evident only after TYB or Percoll sperm processing. The study could not confirm the finding of significant decreases in motility after heat treatment of sperm derived from infertile males. The mechanism for heat-induced hyperactivation did not involve membrane integrity or the sperm acrosome, although an involvement of heat shock proteins was postulated. Interestingly, there were no pregnancies when sperm did not exhibit heat-induced hyperactivation.