J. Corselli

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.

Addition of eosin to the aniline blue assay to enhance detection of immature sperm histones

OBJECTIVES To compare the aniline blue assay with and without eosin, and to correlate the results with pregnancy outcome after intracytoplasmic sperm injection (ICSI) procedure. DESIGN A retrospective study. SETTING University-based fertility center. PATIENT(S) One hundred thirty infertile patients. INTERVENTION(S) Left-over washed sperm after each ICSI procedure were fixed on glass slides and stained with aniline blue with and without eosin. MAIN OUTCOME MEASURE(S) Chromatin condensation, pregnancy, and age. RESULT(S) Percentage chromatin condensation assessed by aniline blue-eosin was higher compared with standard aniline blue (72.4 +/- 2.4% vs. 64.0 +/- 2.4% [mean +/- SEM]). Chromatin condensation was higher in pregnant (86.6 +/- 0.9%) versus nonpregnant (80.9 +/- 2.1%) women age 35 years or more. In younger women, chromatin condensation was not correlated with pregnancy outcome. There was no correlation between chromatin condensation and ICSI fertilization or male age. CONCLUSION(S) Adding eosin counterstain to aniline blue improved assessment of chromatin condensation, suggesting that the standard assay underestimated chromatin condensation. The association between chromatin condensation and pregnancy in older but not younger women suggests that oocytes of younger women had the capacity to compensate for the immature sperm shortcomings.

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.

Enhanced fertility after heat-induced hyperactivation

OBJECTIVE To determine sperm hyperactivation, kinematic parameters, and fertilizing capacity after pretreating sperm at 40 degrees C for 4 hours. DESIGN Prospective study involving pooled donor sperm that were colloid washed and incubated at either 23 degrees C (control) or 40 degrees C (heat-treated) for 4 hours as pretreatment. After incubation, analyses were performed with a computer-assisted sperm analyzer, whereas separate portions of sperm were evaluated with the sperm penetration assay at 37 degrees C. SETTING Clinical and academic research environment. PATIENT(S) Cryopreserved-thawed sperm from different donors (n = 5). MAIN OUTCOME MEASURE(S) Sperm kinematic and fertilizing parameters. RESULT(S) Heat pretreatment of sperm resulted in over 22 times higher hyperactive motility at hour 4 compared with the control. The other kinematic parameters were also different. The heat-pretreated sperm group had a significantly higher percent penetration of zona-free oocytes with more swollen sperm heads per oocyte and enhanced sperm binding. CONCLUSION(S) The results showed that hyperactivation was induced by pretreatment of sperm with 40 degrees C heat, suggesting the involvement of heat factors in hyperactivation. The fertilizing capacity of sperm may be improved by the mild heat pretreatment when marked by the presence of heat-induced hyperactivation.

Correlation Between Intact Sperm Acrosome Assessed Using the Spermac Stain and Sperm Fertilizing Capacity

Accurate determination of sperm acrosomal status is important in fertility studies. The objective was to correlate the percentage of intact acrosome assessed using the new Spermac stain with the capacity of sperm to fertilize oocytes. Sperm specimens were processed either by centrifuge wash, 48:95 Percoll gradient or test yolk buffer (TYB) wash, and tested using the zona-free hamster oocyte assay. The results indicated a correlation between the percentage of sperm with intact acrosome reaction and the percentage of sperm penetrating the oocytes in the TYB-washed group. The data suggest the usefulness of the Spermac stain for assessing the acrosomal status and in predicting the fertilizing capacity of the sperm.

White blood cells in semen affect hyperactivation but not sperm membrane integrity in the head and tail regions

The presence of high numbers of peroxidase-positive PML in ejaculated semen significantly reduced sperm HA, an important step leading to sperm capacitation. Sperm membranes at both the head and tail regions, as assessed by the hypo-osmotic viability parameter and the hypo-osmotic sperm swelling test, respectively, were not affected by peroxidase-containing leukocytes. Sperm motility was not affected, but sperm curvilinear and straight line velocity parameters were reduced in the presence of high concentrations of leukocytes in the ejaculate. The results suggested that the effect of leukocytes on sperm was through a reduction in sperm hyperactive motility but not through alterations in the sperm head and tail membranes.

Assessment of sperm for cryopreservation using the hypoosmotic viability test

In summary, the hypoosmotic viability parameter was significantly correlated with the outcome of the thawed sperm motility. The prefreeze supravital staining for sperm viability and the hypoosmotic sperm swelling test were not predictive of the thawed sperm total motility. The hypoosmotic viability parameter was not correlated to the postwarmed sperm motility after refrigeration. The results indicated that the integrity of the sperm membranes at the head were more important than the tail membrane.

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

AbstractPurpose: 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.

Superovulation using recombinant human FSH and ultrasound-guided transabdominal follicular aspiration in baboon (Papio anubis).

The response of baboon females to a modified human ovarian stimulation protocol incorporating start of pituitary suppression in the luteal phase of the cycle with a GnRH agonist (GnRHa) and recombinant human FSH (rhFSH) was studied. A long-acting GnRHa implant supplying goserelin acetate was administered s.c. to six adult female baboons experiencing regular menstrual cycles (33-34 days) on days 22-24 of the cycle. Follicular development was monitored by transabdominal ultrasonography and serum levels of E2 and progesterone (P4) and rhFSH were determined by ELISA. Menses occurred 9-10 days after GnRHa administration. Daily i.m. administration of 75 IU rhFSH commenced 9-10 days after menses and continued for 9-10 days. When most follicles were > or =5mm diameter and serum E2 had reached its maximum level, 2000 IU hCG was administered i.m. to induce follicle maturation. Transabdominal ultrasound-guided follicular aspiration of follicles > or =2mm diameter was performed 30-34h after hCG administration. One baboon did not show an adequate response to rhFSH stimulation. This animal did not receive further treatment and no data for it are presented. The number of follicles aspirated was 21+/-4 and 17.2+/-3.8 oocytes were recovered per animal with an average recovery rate of 82% (86/105). The number of oocytes collected from five animals were 14, 21, 16, 15, and 20 (n=86). Most of the oocytes recovered were in metaphase II and 3h after recovery 91% (78/86) were considered suitable for in vitro fertilization. It was concluded that recombinant human FSH can successfully induce follicular recruitment and oocyte maturation in baboon females during pituitary suppression with a GnRHa

Luteal phase serum cell-free DNA as a marker of failed pregnancy after assisted reproductive technology

Purpose: DNA-damaging factors have been reported in patients that failed to achieve pregnancy after assisted reproductive technologies (ART). The hypothesis was that increased circulating cell-free DNA released by damaged cells could predict unfavorable conditions leading to failed ART treatment. The objective was to compare the relative concentrations of cell-free DNA in the luteal phase sera of nonpregnant versus pregnant patients.Methods: Frozen-thawed sera (30 IVF cases) were obtained 1 week after embryo transfer. There were 16 pregnant and 14 nonpregnant cases and controls consisting of male sera (n = 8 cases). Modified isocratic capillary electrophoresis was performed and the images analyzed for cell-free DNA.Results: Circulating cell-free DNA were identified in the sera of all patients. The serum concentrations of high (12 kb) and low (1 kb) molecular weight cell-free DNA were similar for both nonpregnant and pregnant patients. Male control sera had higher cell-free DNA concentrations compared with females. Evaluation of sera from a control case showed no fluctuations in cell-free DNA concentrations throughout specific days of the menstrual cycle.Conclusions: The results do not support the use of the luteal phase cell-free DNA concentration as a marker for failed pregnancies. The equal concentrations of high and low molecular weight cell-free DNA and ladder band-like gel patterns suggested cell apoptosis as the source of DNA.