Kevin Coetzee

Stepwise regression analysis to study male and female factors impacting on pregnancy rate in an intrauterine insemination programme

Summary. The aim of this study was to evaluate the impact of male and female factors on the pregnancy rate in an intrauterine insemination (IUI) programme. Data on 522 cycles were retrospectively studied. All patients 39 years or younger were included in the study where data were available on male and female diagnosis, as well as on ovulation induction methodology. Regression analysis was possible on 495 cycles to study different factors affecting the pregnancy rate per treatment cycle. Logistic regression identified variables which were related to outcome and were subsequently incorporated into a statistical model. The number of follicles was found to have a linear association with the risk ratio (chance) of pregnancy. The age of the woman was also found to have a linear (negative) association with pregnancy. The percentage motility and percentage normal morphology (by strict criteria) of spermatozoa in the fresh ejaculate were the male factors that significantly and independently predicted the outcome. Percentage motility ≥ 50 was associated with a risk ratio of pregnancy of 2.95 compared to percentage motility < 50. Percentage normal sperm morphology > 14% was associated with a risk ratio of pregnancy of 1.8 compared to percentage normal morphology ≤ 14%. Female patients with idiopathic infertility were divided into three groups according to normal sperm morphology. The pregnancy rate per cycle was 2.63% (1/38) for the P (poor) pattern group (0–4% normal forms), 11.4% (17/149) for the G (good) pattern group (5–14%), and 24% (18/75) for the N (normal) pattern group (> 14% normal forms). A female diagnosis of endometriosis or tubal factor impacted negatively on the probability of pregnancy (risk ratio of 0.17), compared with other female diagnoses. Male and female factors contribute to pregnancy outcome, but the clinician can influence prognosis by increasing the number of follicles, especially in severe male factor cases.

Perinatal outcomes after fresh versus vitrified-warmed blastocyst transfer: retrospective analysis

OBJECTIVE To investigate the possible effect of controlled ovarian stimulation on the perinatal outcomes of assisted reproductive technology pregnancies, by comparing the outcomes from fresh ET with frozen ET (FET) with blastocysts of similar quality. DESIGN Retrospective observational study. SETTING Private fertility center. PATIENT(S) Seven hundred eighty-four fresh transfers and 382 vitrified-warmed double blastocyst transfers. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Miscarriage, perinatal mortality, preterm delivery, live birth, live-birth weights, and gestational age of live births. RESULT(S) FET resulted in higher implantation rates (51.5% vs. 40.6%), higher live-birth rates per transfer (56.8% vs. 44.3%), and lower ectopic pregnancy rates (0.32% vs. 1.80%). FET pregnancies also had higher day 14 βhCG levels per implantation (148.2 vs. 176.2 IU/L) and higher infant birth weights (singletons Δ109.4 g, twins Δ124 g). Female infants benefitted the most in terms of birth weight. Miscarriage, premature delivery, perinatal morbidity, and live birth per pregnancy were all nonsignificantly different between fresh ET and FET. CONCLUSION(S) Clinically significant differences between the peri-implantation and perinatal outcomes of fresh ET and FET suggest better endometrial receptivity and placentation in FET cycles.

Repeatability and variance analysis on multiple computer-assisted (IVOS*) sperm morphology readings

The repeatability of the Hamilton Thorne Research IVOS (version 10) semen analyser (dimension specific software, version 3) in the evaluation of sperm morphology according to strict criteria was investigated in this study. The repeat measures investigated were cell‐cell (300 cells, 3 X each), intraslide (20 slides, 3 X each) and interslide (30 samples, 3 slides each), and their normal sperm morphology outcomes were recorded. Semen samples with varying normal sperm morphology percentages were obtained and sperm morphology slides prepared. The slides were stained with Diff‐Quik stain. Agreements between evaluations were determined using the κ statistic and average coefficients of variation. The predictive probability for an abnormal cell given a prior abnormal cell outcome was 91%, and 89% for a similar prediction of a normal cell. The predictive probabilities for an abnormal or a normal cell given two prior abnormal or two prior normal cell outcomes were 95% and 94%, respectively. No significant bias was obtained between the repeat probabilities for normal and abnormal sperm cells. The average coefficients of variation for the intraslide trial were 9.73% and 8.30% when 100 and 200 sperm cells were evaluated, respectively. The average coefficient of variation for the interslide trial was 15.39%. The technical importance of good sample and slide preparation technique has once again been highlighted by this study. A uniform (spatial homogeneity), high concentration (5–10 cells per computer screen) smear must be made and the cells stained with optimal intensity (maximum contrast). In a trial in which 2000 cells were evaluated, 19 objects (0.95%) were identified as spermatozoa, but were debris. The automated semen analysing system (IVOS) used in this study was shown to maintain a level of repeatability, precision and accuracy acceptable for the application of the system in a routine semen analysis situation.

Slide Preparation and Staining Procedures for Reliable Results Using Computerized Morphology

The purpose of this study was to standardize slide preparation and staining procedures to improve the efficiency and effectivity of the IVOS system on normal sperm morphology readings with regard to the strict criteria. Semen samples from patients attending the Reproductive Biology Unit, Tygerberg Hospital, were used. In experiment 1, five different Diff-Quik staining procedures, including the standard procedure, were evaluated on each of 22 patients and the effect of slide preparation within 1 h or more than 5 h after collection and the effect of immediate fixation versus fixation after 24 h were observed. In experiment 2, the manual evaluation time per slide (n = 20) by two technicians was compared with the time taken by computer. In experiment 1 the median % normal for the 5 different staining procedures was 6, 6.5, 9.5, 8.5, and 5.5%. No significant difference was found between the different staining procedures (p = .60, nonparametric Friedman test). In experiment 2 the mean time for manual assessment by two technicians was 3 min:6 s and 3 min:53 s per slide as compared to 4 min:39 s by computer. For experiment 1, slides can be prepared immediately or after 5 h. Fixation time also does not interfere with the computers ability to identify normal forms. For experiment 2, the IVOS system is competitive regarding assessment time. Standardization of optimum staining procedures is important to ensure repeatability and comparability. Therefore, slides should be prepared immediately after liquefaction and fixed immediately after air drying.

Assessment of interlaboratory and intralaboratory sperm morphology readings with the use of a Hamilton Thorne Research integrated visual optical system semen analyzer

OBJECTIVE To evaluate the level of variance produced in a multicenter study with the use of a computer-assisted sperm morphology analyzer. DESIGN A multicenter, prospective, blinded study. SETTING Assisted reproduction research laboratories. PATIENT(S) Semen samples produced for assisted reproductive procedures. INTERVENTION(S) Hamilton Thorne Research (Beverly, MA) integrated visual optical system semen analyzers were used at five different centers to evaluate the same set of 30 slides that were prepared and numerically coded at Tygerberg Hospital in Tygerberg, South Africa. MAIN OUTCOME MEASURE(S) The percentage of normal sperm. RESULT(S) Interlaboratory coefficients of variation (CVs) ranged between 16.31% and 23.09%. One of the participating laboratories produced an approximately 14% (-6.5-7.7) limits of agreement analysis, with a CV of 11.36%, for its duplicate readings. The use of a 10% normal sperm morphology cutoff point to determine discordance levels produced rates ranging between 10% and 23.3% for the interlaboratory and intralaboratory readings. This level of discordance equates with < or = 7 of the corresponding readings from two laboratories falling into a different normal sperm morphology group (< or = 10% or >10%). CONCLUSION(S) The magnitudes of variation produced by the readings performed in our study reached the same level as for the manual evaluation of sperm morphology. A < 10% CV can be obtained if the correct quality control measures are implemented.

Usefulness of Sperm Penetration Assay in Fertility Predictions

The competence of the sperm penetration assay (SPA) to predict male fertility, as determined by normal sperm morphology and the fertilizing potential, as shown by human in vitro fertilization (IVF), was investigated. A significant correlation was obtained between normal sperm morphology and the SPA (phi = 0.623). A weaker correlation was however obtained with human IVF (phi = 0.397). Notwithstanding this weak association, a positive SPA (greater than 10%) was highly predictive (95%) of human IVF success. In contrast, a negative SPA (less than or equal to 10%) was associated with a high rate of false-negatives (65%). The SPA does however warn that a male factor may be present, as the mean fertilization rate of this group of patients was markedly reduced. The preincubation period for the spermatozoa did not play a major role in the predictive ability of a SPA outcome.

VALIDATION OF A NEW DISPOSABLE COUNTING CHAMBER

In a routine prospective study, 3 different counting chambers were compared for manual and computerassisted evaluations. Swim-up samples were used so as to remove all debris and other cells that may contaminate the evaluation process when using a semen analyzer. The Makler concentration determinations ( n = 20) were, on average, approximately 20 2 10 6 cells/mL higher compared to the corresponding 20 hemocytometer counts. The mean differences between the Leja chambers and the hemocytometer counts ( n = 320) were only around 1 2 10 6 cells/mL, with coefficients of variation around 20%. The Leja chambers for both manual and the computer-assisted sperm concentration determinations provided consistent and accurate data on sperm concentration.

Clinical value of using an automated sperm morphology analyzer (IVOS)

OBJECTIVE To determine the clinical value of automated normal sperm morphology outcomes. DESIGN Prospective clinical study. SETTING Clinical and research assisted reproduction laboratory. PATIENT(S) Two hundred seven GIFT cycles. INTERVENTION(S) The wife was induced to superovulate, laparoscopically aspirated, and the gametes were transferred laparoscopically. The husbands sperm morphology was evaluated with use of a sperm morphology analyzer using the strict criteria classification system. MAIN OUTCOME MEASURE(S) Normal sperm morphology, IVF, and pregnancy outcomes. RESULT(S) The logistic regression model showed that normal sperm morphology was significantly associated with fertilization in vitro, as dependent (age) and independent variables. Analyzing the fertilization rates across the 5% normal sperm morphology cutoff point, a fertilization rate of 39.39% (< or = 5%) compared with 62.92% (>5%) was obtained. The logistic regression model showed that normal sperm morphology was also a significant predictor of pregnancy when allowing for the number of oocytes transferred and female age. Analyzing the pregnancy rates across the 5% normal sperm morphology cutoff point, pregnancy rates of 15.15% (< or = 5%) and 37.36% (>5%) were obtained. CONCLUSION(S) Normal sperm morphology as evaluated by the automated semen analyzer (IVOS) was shown to adhere to the same fertility cutoff point (5%), as determined by the manual evaluation of sperm morphology. Automated normal sperm morphology outcomes also were found to be significant predictors of IVF and pregnancy in a GIFT program.

Comparison of motility characteristics and normal sperm morphology of human semen samples separated by Percoll density gradient centrifugation

The aim of this study was twofold: to investigate the ability of Percoll gradient centrifugation (52, 68, 84%) to fractionate semen samples according to motility quality and percentage normal morphology and to determine whether there is an association between sperm motility quality and percentage normal morphology. Sperm motility was evaluated using a Hamilton Thorn analyzer and normal sperm morphology was manually assessed according to the strict criteria (< or = 4, 5-14, and >14%). The majority of motility parameters and the percentage normal morphology were found to be significantly improved in the 84% Percoll fraction. The greatest effect was on the < or = 4% group, shifting the mean normal morphology percentage from 2.6 to 5.2%. Curvilinear velocity (VCL) and average path velocity (VAP) were the only two motility parameters that were significantly associated with the percentage normal morphology. Using a combined VCL, VAP vector the >14% group was found to have a significantly different value as compared to the 5-14 and < or = 4% groups. Percoll (discontinuous) gradient centrifugation can therefore play a significant role in the improvement of semen samples for use in assisted reproduction procedures. The VCL, VAP vector identified may also serve as an additional tool in the prediction of the fertility potential of sperm samples.

Glass wool filter preparation of cryopreserved spermatozoa

Summary: Oligozoospermic and asthenozoospermic semen ejaculates, as well as cryopreserved sperm samples prepared by the wash and swim‐up procedure often result in unsatisfactory sperm recovery rates. In this study the glass wool filter and the wash and swim‐up preparation procedures were compared on the basis of their ‘effective’ (number of live sperm per millilitre) recovery rates. The glass wool filter procedure consistantly produced significantly (P = 0.0002) higher viable sperm concentrations, making it the preferred method for the preparation of cryopreserved sperm to be used in assisted reproduction techniques. The use of this preparation procedure has also been shown to have no adverse affect on the fertilizing potential of human spermatozoa in our unit.