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Biomedical subjects

W C Ford

Publications and source records attributed to W C Ford.

At least 37 records · Page 2Linked to original sources
PubMed1997

Reactive oxygen species in human sperm suspensions: production by leukocytes and the generation of NADPH to protect sperm against their effects.

In men from couples consulting for infertility, 72% of washed sperm preparations produced detectable amounts of reactive oxygen species (ROS) compared to only 25% of preparations from a population of fertile donors. High ROS production was associated with oligozoospermia and poor sperm function. The rate of ROS production was closely correlated with the concentration of leukocytes (r = 0.826) and the degrees of stimulation by N-formyl-methionyl-leucyl-phenyl alanine (NFMLP) and by phorbol 12-myristate 13 acetate (PMA) were similar (r = 0.923). The removal of leukocytes with 'Dynabeads' either abolished or substantially decreased ROS production but in a few cases ROS production that could be stimulated by PMA but not NFMLP was observed in leukocyte-free preparations. When sperm preparations which produced ROS were incubated under 95% O2 there was a rapid 40% decrease in the number of sperm that could be stimulated to acrosome react although the acrosome reaction was unaffected by incubation under 95% N2 for up to 6 h. The harmful effect of oxygen was not seen in preparations that produced no ROS and could be prevented by removing leukocytes from the suspension or by adding superoxide dismutase and catalase. We conclude that leukocytes are the predominant source of ROS in human sperm preparations and that the ROS they produce are harmful to sperm. On the other hand these data confirm that highly purified sperm can produce ROS albeit in smaller amounts. We have demonstrated that flux through the pentose phosphate pathway (PPP) in purified sperm preparations increases in response to oxidative stress. This is required to make reduced glutathione available for glutathione peroxidase and we suggest that measurement of PPP flux provides an index of the capacity of glutathione peroxidase to protect sperm against oxidation.

Humans↗
PubMed1996

Randomized controlled trial of cervical cap with intracervical reservoir versus standard intracervical injection to inseminate cryopreserved donor semen.

A prospective controlled study of donor insemination without sperm preparation or ovarian stimulation was performed to compare the use of a cervical cap incorporating an intracervical reservoir with a standard intracervical injection technique to inseminate 0.5 ml cryopreserved semen. Treatments were alternated in successive cycles in each patient after initial randomized selection. A total of 198 patients had 635 treatment cycles (median 3, range 1-7), 309 with reservoir and 326 by standard injection. A total of 56 women became pregnant, 24 (7.8% per cycle) with the reservoir and 32 (9.8% per cycle) by injection. There were no significant differences between the pregnancy rates per cycle overall or cycle-specific cumulative rates calculated using the life-table method. There were no significant differences in age, parity, baseline gonadotrophin measurements, mid-luteal serum progesterone concentrations, frequency of adverse fertility factors in the woman or her partner's cause of infertility between women who conceived and those who failed to conceive. We conclude that use of a cervical reservoir and cap for donor insemination does not offer any advantage over standard intracervical insemination.

Adult↗
PubMed1996

The indirect immunobead test for seminal antisperm antibodies and fertilization rates at in-vitro fertilization.

A series of 183 patients with positive indirect immunobead tests on semen was studied to determine the correlation in semen between specific antibody types, binding sites, antibody concentration, and fertilizing ability. IgM was present in only 44 ejaculates and was present in sufficient quantity to cause significant binding to immunobeads (i.e. >20% of motile donor spermatozoa) in only three of them. There was no correlation between the percentages of motile donor spermatozoa that bound IgA and IgG immunobeads but the two classes of beads generally bound to the same region of the spermatozoa. A total of 63 couples went on to attempt in-vitro fertilization (IVF) treatment, all with mature eggs recovered. Of these mature eggs, 44% were fertilized and cleaved normally in comparison to 68% in a group of patients with tubal disease. Fertilization rates in individuals followed a bimodal distribution with a substantial number of couples experiencing zero or very poor rates (0-20%), the mode for the remainder lying between 60 and 80%. The fertilization rate tended to decrease as the amount of antibody increased. The percentage of donor spermatozoa that bound to immunobeads, taken as the greater of IgA and IgG, was selected by logistic regression as a significant predictor of poor fertilization (rate <=25%). The predictive power of the equation was improved by including the motile normal sperm concentration but the equation could only account for a small proportion of the total variation in fertilization rate. The presence of antibodies to the sperm head was highly correlated with the antibody concentration but was not selected as a predictor of fertilization. We conclude that the nature of the antigen against which the seminal antisperm antibody is directed may be as important as the antibody concentration in affecting sperm function. There seems to be little practical value in measuring IgM in seminal plasma.

Adult↗
PubMed1995

Intracellular calcium measurements in individual human sperm demonstrate that the majority can respond to progesterone.

OBJECTIVE: To determine if human spermatozoa could be immobilized and intracellular calcium measurements made on individual cells to measure what proportion can respond to P. DESIGN: Spermatozoa were loaded with Fura 2 (Sigma Chemical Co., Poole, Dorest, United Kingdom) and suspended in 10% gelatin at 37 degrees C. A thin layer of the suspension was cooled to room temperature (20 degrees C to 25 degrees C) and [Ca2+]i was measured with a fluorescence microscope equipped with dual wavelength excitation and an image analysis system. SETTING: University-based laboratory. PARTICIPANTS: Semen was obtained from four fertile donors to a donor insemination program. INTERVENTIONS: None. MAIN OUTCOME MEASURES: [Ca2+]i was calculated from the ratio of Fura 2 fluorescence excited at 340 nm and that excited at 366 nm. RESULTS: One hundred six of 114 sperm examined (93%) demonstrated a significant response to P but the size and duration of the response was variable. CONCLUSION: These data demonstrate that most sperm can respond to P.

Calcium↗
PubMed1994

Effects of cryopreservation on the intracellular calcium concentration of human spermatozoa and its response to progesterone.

The intracellular free calcium concentration [Ca2+]i of sperm from 23 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura-2. Spermatozoa were treated with 3.18 microM progesterone so that the regulation of [Ca2+]i in a dynamic situation could be studied. [Ca2+]i (nM) was 290 +/- 13 in fresh spermatozoa vs. 550 +/- 26 in cryopreserved samples (mean +/- S.E.M. P < 0.0001 paired t-test). Progesterone at a dose of 3.18 microM stimulated a large and rapid increase in [Ca2+]i to a peak value > 1 microM after 10-20 seconds. [Ca2+]i then declined to a slightly raised basal level over the next 30-40 seconds. This phenomenon occurred in all the fresh samples, but about half the frozen thawed samples failed to respond. The peak [Ca2+] attained by frozen samples which did respond after the addition of progesterone was similar to that observed with fresh sperm. The calcium channel blocker verapamil (200 microM) completely inhibited the transient rise in [Ca2+]i produced by progesterone, but 100 microM verapamil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca2+]i in human spermatozoa and (2) damage to the plasma membrane during cryopreservation may result in the loss of the progesterone receptor. Both factors may contribute to the loss of fertility after cryopreservation.

Calcium↗
PubMed1994

The effect of halving the standard dose of cryopreserved semen for donor insemination: a controlled study of conception rates.

Employing a common standard technique of intra-cervical insemination from straws of cryopreserved donor semen, a volume of 0.25 ml of 0.5 ml was inseminated in alternate cycles to determine if the lower volume could be used without a decrease in the conception rate. A total of 177 women were recruited and received a median of four cycles of treatment. Of these, 90 women became pregnant, 47 with 0.5 ml and 43 with 0.25 ml inseminations. The conception rates were identical for both volumes in the first nine cycles of treatment and the cumulative rates were 57.7 and 59.3%, respectively. Subsequently more pregnancies were achieved with 0.5 ml than 0.25 ml semen (nine pregnancies in 73 further cycles versus three pregnancies in 68 cycles, respectively), although the difference was not statistically significant. There were no significant differences in the women's ages, luteinizing hormone, follicle stimulating hormone, progesterone, mucus quality, mucus pH, parity or partner's diagnosis between those women who became pregnant and those who failed to conceive with either insemination dose. We conclude that the volume of semen inseminated into the cervical canal without a cervical cap can be decreased to 0.25 ml without an adverse effect on the conception rate at least in the first 9 months of treatment. This will allow more effective use to be made of valuable screened and quarantined cryopreserved semen.

Adult↗
PubMed1994

Adenosine triphosphate and motility characteristics of fresh and cryopreserved human spermatozoa.

The concentration of ATP and the motility of human spermatozoa was measured in fresh and cryopreserved cells from the same 15 ejaculates. No coherent picture of the relationship between motility and ATP concentration emerged in whole semen or in spermatozoa washed by repeated centrifugation and resuspension in Biggers Whitten and Whittingham medium. This may have been due to the presence of dead spermatozoa and contaminating cells. After preparation on a Percoll gradient, the ATP concentration in fresh and cryopreserved spermatozoa was the same (6 +/- 0.7 nmol/10(8) spermatozoa) but 85 +/- 2.5% of the fresh spermatozoa were progressively motile with an average path velocity of 55 +/- 3.5 microns/s compared to corresponding values of 33 +/- 5.3% and 44 +/- 3.4 microns/s in frozen/thawed spermatozoa. This suggests that the poor motility of cryopreserved spermatozoa does not result from deficient ATP production. No relationship was found between ATP concentration and the ability of motile spermatozoa in the ejaculate to survive freezing.

Adenosine Triphosphate↗
PubMed1994

Pentoxifylline acts synergistically with A23187 to increase the penetration of zona-free hamster oocytes by cryopreserved human spermatozoa.

The number of cryopreserved human spermatozoa which penetrated zona-free hamster oocytes after stimulation with 2 mumol A23187 per litre was increased by the further addition of 0.6 or 3.6 mmol pentoxifylline per litre. With spermatozoa prepared by washing by repeated centrifugation, the median numbers of sperm heads/egg were 1.9, 7.9 and 10.8 in the presence of 0, 0.6 or 3.6 mmol pentoxifylline per litre, respectively. A similar effect was observed with spermatozoa prepared on a Percoll gradient. As A23187 inhibited sperm motility, and this was exacerbated by pentoxifylline, the increased penetration rate of hamster oocytes cannot be explained by improved sperm motility. The number of spermatozoa stimulated to acrosome react by 2 mumol A23187 per litre was increased 3-fold by 3.6 mmol pentoxifylline per litre and 4-fold by 5 mmol caffeine per litre. These data suggest that cAMP may act synergistically with Ca2+ to stimulate the acrosome reaction. Pentoxifylline may improve the fertility of poor-quality human spermatozoa by enhancing their ability to respond to the Ca2+ signal produced by binding to the zona pellucida.

Acrosome↗
PubMed1994

Relative influence of serum follicle stimulating hormone, age and other factors on ovarian response to gonadotrophin stimulation.

OBJECTIVE: To determine the relative value of the woman's age, basal follicle stimulating hormone (FSH), basal luteinising hormone (LH) and menstrual cycle pattern (all defined prior to treatment) in predicting the ovarian response to gonadotrophin stimulation for in vitro fertilisation. DESIGN: Open, descriptive cohort study. PATIENTS: One hundred and seventy-one consecutive ovulatory women undergoing their first cycle of in vitro fertilisation with ovarian stimulation after pituitary desensitisation. INTERVENTIONS: Measurement of basal (early follicular phase) and down-regulated concentrations of serum FSH and LH, and calculation of age at treatment and of median length of menstrual cycles and range of variation prior to treatment. MAIN OUTCOME MEASURES: Duration and amount of gonadotrophin stimulation to achieve follicular maturity, number of mature follicles, peak serum oestradiol concentration and number of mature oocytes, were compared between banded values of variables studied. RESULTS: Increasing basal FSH concentrations were significantly associated with reducing oestradiol levels, numbers of mature follicles and oocyte yield even when the FSH levels were still within the normal range though above average (6 to 8.9 i.u./l). There were similar but weaker associations with FSH levels after down-regulation. There were also significant differences between women over and under 40 years old in their oestradiol levels, numbers of follicles and of oocytes. Analysis of variance showed significant independent association of basal FSH with both oestradiol and numbers of oocytes, but not of age, menstrual pattern, or serum LH concentrations. CONCLUSION: Serum FSH is more sensitive than the woman's age in determining her ovarian responsiveness to stimulation. LH concentrations and menstrual cycle patterns are unhelpful.

Adult↗
PubMed1993

Effects of cryopreservation on the human sperm acrosome and its response to A23187.

The proportion of human spermatozoa from 28 ejaculates to lose their acrosomes during cryopreservation was measured and correlated with the number that became immotile or lost the integrity of their plasma membrane. The ability of washed spermatozoa to acrosome react in response to A23187 before and after cryopreservation was compared. Motility was assessed by time-lapse photography; intact acrosomes were stained with fluorescein conjugated Pisum sativum agglutinin and dead spermatozoa were stained with bisbenzimide (H33258). Twenty-four per cent of spermatozoa lost their acrosomes during freezing and thawing, but the number that did so was not correlated with the number that became immotile or non-viable. Frozen spermatozoa exhibited fewer spontaneous acrosome reactions than did fresh spermatozoa (5 versus 13% after 4 h), but they responded to A23187 in a similar way. Although frozen spermatozoa were significantly more likely to die during the incubation, the data do not suggest that degenerative acrosome loss had a major influence on the results. In the hamster egg test frozen-thawed spermatozoa achieved more penetrations than did fresh spermatozoa when stimulated with 0 or 1 mumol A23187 l-1 but considerably fewer when stimulated with 4 mumol A23187 l-1. The following conclusions were made. First, cryopreservation damage to the acrosome, the plasma membrane and the flagellum can occur independently. Second, acrosome function is maintained after cryopreservation as long as the organelle remains mechanically intact. Third, some spermatozoa that lose their acrosomes during cryopreservation remain viable and can fuse with zona-free hamster eggs.

Acrosome↗
PubMed1992

Smoking and decreased fertilisation rates in vitro.

To examine possible mechanisms for the association between cigarette smoking and reduced fertility, we have measured the concentration of the nicotine metabolite cotinine in ovarian follicular fluid collected at the time of oocyte recovery during treatment for in-vitro fertilisation. In a group of women in whom follicular fluid cotinine could not be detected (limit of accurate measurement 20 ng/ml) 116 oocytes were collected, of which 84 became fertilised (72%), whereas among women with cotinine concentration greater than 20 ng/ml 20/45 (44%) oocytes did so (p < 0.01). The median fertilisation rates for individuals (range 1-8 eggs each) in the high and low cotinine groups were 57% and 75%, respectively (p < 0.05). These findings suggest that infertile women should be advised to stop or reduce smoking generally, and especially before treatment by in-vitro fertilisation.

Adult↗
PubMed1992

Expectations of assisted conception for infertility.

OBJECTIVE: To provide reliable prognostic information for couples seeking assisted conception. DESIGN: Analysis of four years' practice (1988-91). SETTING: Private university service linked with NHS reproductive medicine services. PATIENTS: 804 couples with various causes of subfertility, median duration five years, median age of women 34 years. INTERVENTIONS: 1280 completed cycles: 950 in vitro fertilisation, 144 gamete intrafallopian transfer, and 186 intrauterine insemination and superovulation. MAIN OUTCOME MEASURES: Pregnancy and birth rates per cycle and cumulative pregnancy and take home baby rates per couple. RESULTS: In women under 40 years and men with normal sperm, whatever the cause of infertility, results with in vitro fertilisation improved steadily reaching a pregnancy rate per cycle of 30% (95% confidence interval 26% to 35%) during 1990-1 and birth rate per cycle of 29% (23% to 35%) in 1990. Pregnancy and birth rates for gamete intrafallopian transfer were 36% (28% to 44%) and 26% (17% to 37%) and for intrauterine insemination 18% (12% to 24%) and 16% (10% to 22%). After six cycles cumulative probability of pregnancy was 82% and cumulative take home baby rate 70%. Considering only in vitro fertilisation and gamete intrafallopian transfer after four cycles the pregnancy rate was 78% (66% to 91%). CONCLUSIONS: Conception is less likely in women over 40 and men with sperm dysfunction. For other couples the prognosis for a live birth is at least as good as for fertile couples if they persist with treatment.

Adult↗
PubMed1992

The yield, motility and performance in the hamster egg test of human spermatozoa prepared from cryopreserved semen by four different methods.

Different procedures were investigated for the dilution of human cryopreserved semen and the preparation of an enriched population of motile spermatozoa for assisted reproduction. The dilution of a 0.25 ml straw of cryopreserved human semen by addition of 2.0 ml Ham's F-10 buffer in one step caused a large decrease in the proportion of motile spermatozoa. This was due to osmotic stress because many of the diluted spermatozoa exhibited swollen tails. To a large extent the damage could be avoided by adding the buffer in 0.10-ml aliquots at 30-s intervals. Spermatozoa obtained after such dilution of cryopreserved human semen were subjected to the swim-up procedure, to centrifugation on two-step gradients of Nycodenz or Percoll, or to filtration through glass fibre paper and compared with respect to yield, motility parameters and penetrating ability in the hamster egg test. The swim-up procedure yielded spermatozoa with excellent motility but only 12% of the available motile spermatozoa were recovered. On both Nycodenz and Percoll gradients, greater than 40% of the available motile spermatozoa were recovered and the average velocity of the spermatozoa was not significantly less than for the swim-up technique. When A23187 was used to promote acrosome reactions in the hamster egg test, Percoll-prepared spermatozoa achieved an average of 8.6 decondensed sperm heads/egg compared to 1.9 for Nycodenz and 1.3 for the swim-up procedure. The yield from glass fibre paper filtration was only 12% and the velocity of the spermatozoa and their performance in the hamster egg test was significantly poorer than in all the other methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Controlling the swimming speed of human sperm by varying the incubation temperature and its effect on cervical mucus penetration.

The objective of the present experiments was to study the effect of sperm velocity as a single variable on the ability of sperm to penetrate cervical mucus in a modified Kremer test. Sperm incubated at 13, 22 and 37 degrees C exhibited progressive velocities of 25 +/- 1.7, 40 +/- 2.1 and 56 +/- 2.1 microns sec-1 (mean +/- SEM, n = 6) respectively, but the percentage of progressively motile sperm, their lateral head displacement and the viscoelastic properties of cervical mucus remained comparatively unchanged over this temperature range. The number of sperm which penetrated the mucus and the percentage of successful collisions were correlated strongly with the average velocity of the sperm population (r = 0.82 and r = 0.72 respectively). It is concluded that sperm velocity has an important influence on the penetration of cervical mucus because it governs the frequency of collisions with the mucus interface and is determined by the thrust generated by the flagellum which also determines the ability of the sperm to traverse the mucus interface.

Cervix Uteri↗
PubMed1992

Motility characteristics and membrane integrity of cryopreserved human spermatozoa.

The motility characteristics of washed spermatozoa from 50 normal ejaculates were measured by time-lapse photography, before and after cryopreservation. Plasma membrane integrity was assessed by the hypo-osmotic swelling test and with the supravital fluorescent dye bisbenzimide (H33258). There was a marked decline in the percentage of progressively motile spermatozoa after cryopreservation, the extent varying widely among donors. Results were, however, consistent between different ejaculates from the same individual. The ability of spermatozoa to survive cryopreservation could not be predicted from the properties of the semen beforehand. The mean velocity of the spermatozoa was significantly reduced after freezing, but the lateral head displacement was unaltered. There was a significant reduction in the proportion of spermatozoa with intact plasma membranes after cryopreservation and the results of the hypo-osmotic swelling test and H33258 tests correlated closely. There was no correlation between the declines in the percentage of motile spermatozoa, or intact spermatozoa and the sperm velocity. We conclude that membrane rupture is not the sole cause of loss of motile spermatozoa during freezing and that the decrease in the proportion of motile spermatozoa is caused, at least in part, by a separate process from that responsible for the decrease in the average swimming speed of spermatozoa.

Analysis of Variance↗
PubMed1992

The contribution of the toxicity of a glycerol-egg yolk-citrate cryopreservative to the decline in human sperm motility during cryopreservation.

The contribution of the toxicity of glycerol-egg yolk-citrate (GEYC) cryopreservative medium to the loss of function of human spermatozoa during cryopreservation was determined by investigating the effect of mixing semen with the medium on sperm motility. The percentage of progressively motile spermatozoa, velocity (micron s-1) and lateral head displacement (micron) (mean +/- SEM, n = 28) were 55 +/- 4.1, 47 +/- 2.7, 4.4 +/- 0.2 and 32 +/- 3.8, 40 +/- 2.5, 3.6 +/- 0.25 and 15 +/- 2.5, 28 +/- 1.1, 2.8 +/- 0.15 in suspensions of washed spermatozoa prepared from fresh, GEYC-treated and frozen-thawed semen, respectively. The variables changed only slightly after incubation for 3 h. The toxicity of GEYC did not vary significantly between samples which survived the complete freeze-thaw cycle well or very poorly. The toxicity of GEYC is responsible for about 50% of the loss of progressively motile spermatozoa during the complete cryopreservation process, but has little effect on the quality of motility. Susceptibility to GEYC does not explain observed differences in the ability of semen samples to survive freezing.

Citrates↗
PubMed1991

The effect of A23187 concentration and exposure time on the outcome of the hamster egg penetration test.

We investigated the conditions required to enhance the performance of human sperm in the hamster egg penetration test with the free acid form of A23187. The best performance was observed after stimulation with 2 microM A23187 for 1 h when the median penetration rate with sperm from fertile donors was 100% of eggs with 5.8 decondensed sperm heads/egg. Extending the stimulation period with 2 microM A23187 to 2 or 3 h, resulted in a progressive decrease in the penetration rate. In the absence of A23187, the penetration rate was lower (0.7 decondensed sperm heads/egg after 1 h) but increased with stimulation time. A similar picture was observed with sperm from patients taken for an IVF programme. For a pool of cryopreserved semen, the coefficient of variation of the penetration rate after stimulation with 2 microM A23187 for 1 h, expressed as decondensed sperm heads/egg, was 11% within and 20% between assays. There was no correlation between the outcome of the hamster egg penetration test and the percentage motility, velocity or lateral head displacement of the sperm measured after the same stimulation regime. However, in IVF patients the initial velocity and lateral head displacement of the sperm (zero time) were correlated with the best result from the hamster egg penetration test (r = 0.62 and 0.57 respectively). No motility changes characteristic of capacitation were detected. We conclude that stimulation with 2 microM A23187 (free acid) for 1 h prior to the addition of the zona free hamster eggs can produce a high penetration rate with fertile samples and provides a convenient and robust protocol for the assay. However, when carried out in this way the test does not assess the ability of the sperm to capacitate.

Acrosome↗