John D. Jacobson

A population-based study of maternal and perinatal outcome in patients with gestational diabetes

A prospective population-based study of gestational diabetes mellitus was done with 2272 patients to determine perinatal and maternal outcomes. A large data base was collected on all patients. Patients with gestational diabetes mellitus were older, shorter, heavier, and had more children than did the control group. The higher cesarean section rate in the patients with gestational diabetes mellitus was explained by their increased rate of repeat cesarean section compared with control patients. This was associated with increased infectious complications. Other maternal complication rates were similar in the two groups. Acceptable glucose control did not normalize birth weight percentiles in patients with gestational diabetes mellitus. Maternal weight at delivery was the only significant predictor of birth weight percentile in the group with gestational diabetes mellitus. Plasma glucose levels were a poor predictor of birth weight percentile. Factors associated with maternal obesity in well-controlled gestational diabetes mellitus may be more significant than glucose control in the development of large-for-gestational-age infants.

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.

Modified isocratic capillary electrophoresis detection of cell-free DNA in semen.

Purpose: The objectives were: i) to analyze semen for the presence of cell-free DNA and ii) to determine the association between sperm parameters and cell-free DNA.Methods: Cell-free DNA in semen (N=25 cases) were detected using the modified capillary gel electrophoresis (CE) procedure. SYBR-Gold was used to stain high (12 Kb) and low (1 Kb) molecular weight DNA fragments and the images analyzed.Results: The quantity of low-molecular weight cell-free DNA was positively correlated to rapid progression, curvilinear velocity (>40 μ/s), normal strict morphology and capacitation index. High-molecular weight cell-free DNA intensity index was negatively correlated to post-wash hyperactivation. Sperm concentration was not related to cell-free DNA quantity. The sperm freezing process did not increase cell-free DNA but reduced the more labile low-molecular weight cell-free DNA.Conclusions: Cell-free DNA present in semen was correlated to important sperm parameters linked to normal sperm function. The data suggested the possible use of cell-free DNA as a marker of semen quality. This study reports on the novel finding of cell-free DNA released along with sperm during each ejaculation.

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.

Sperm Kinetics and Morphology Before and After Fractionation on Discontinuous Percoll Gradient for Sex Preselection: Computerized Analyses

The multiple-layer discontinuous Percoll density gradient centrifugation procedure is being used for gender selection and several reports suggested separation efficiencies of over 77%. The mechanism involved in the separation of X- and Y-bearing sperm using this method seems to be the difference in sperm head dimensions or motility but supporting data are inconsistent. The specific aims of the study were to evaluate the head dimensions of sperm at the upper and lower fractions after the 8-layer Percoll gradient procedure for sex preselection and to ascertain the kinematic parameters and tail lengths of sperm derived from the 2 separate Percoll fractions. Sperm cells were obtained from thawed donor specimens (N = 20) and were layered on top of the 8-layer discontinuous Percoll gradient, which ranged from 34 to 85% in increments of 7%. After centrifugation, the resuspended sperm cells derived from the upper and lower fractions of the Percoll gradient were analyzed on the Hamilton Thorn HTM-C analyzer for differences in sperm motility patterns and sperm head dimensions. Aliquots of sperm from the 2 fractions were fixed and stained using the Spermac stain, and the lengths of each sperm tail (N = 600) were measured on the HTM-C analyzer. Sperm derived from the bottom Percoll (X) fraction had a threefold higher (p < .05) percent motility when compared with sperm from the top (Y) fraction. Sperm derived from the bottom (X) fraction maintained the higher percentage motility after 24 h of incubation. The percent total progression, rapid progression, and hyperactivation were also significantly higher (p < .05) in sperm from the bottom (X) fraction. Similarly, the curvilinear (Vcl), average path (Vap) and straight line (Vsl) velocities were significantly faster in sperm (p < .05) from the bottom (X) fraction. In contrast, the percent linearity and straightness were significantly (p < .05) higher for the top (Y) fraction. Sperm from the bottom (X) fraction have shorter (p < .07) tail length (1.6% difference) when compared with sperm from the top (Y) fraction. Although the dimensions of the sperm head from the bottom (X) fraction were numerically greater than top fraction sperm, they were not significant (p > .05). The results suggest that bottom (X) fraction sperm derived from the 8-layer discontinuous Percoll gradient for sex preselection have higher motility, progression, and hyperactivation when compared with top (Y) fraction sperm. The bottom (X) fraction sperm have greater longevity in motility and have shorter tails, supporting earlier hypotheses of sex differences in sperm parameters. However, the present data do not support observations of differences in sperm head dimensions in sperm processed for sex preselection, and an inference of a larger sperm due to more chromosome material originating from the X chromosome cannot be made.

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.