T. F. Kruger

Urogenital inflammation: changes of leucocytes and ROS

Summary. The presence of excess leucocytes in the semen has been associated with male infertility. According to the WHO, concentrations of more than 106u2003leucocytesu2003ml−1 are considered as leucocytospermia, indicating genital tract infections. Up to now, no consensus has been achieved on how leucocytes should be quantified in semen. Using the peroxidase staining and monoclonal antibodies to CD15, CD45 and CD68, we found significant differences between the detection methods. Only 47.4% of the semen samples that were assessed as leucocytospermic by CD45 were identified as such by peroxidase staining. The concentration of peroxidase‐positive cells was significantly correlated with polymorphonuclear granulocyte (PMN) elastase (Pu2003<u20030.0001). However, a negative correlation of peroxidase‐positive cells with the sperm concentration was only found in oligozoospermic patients (Pu2003<u20030.0001). Moreover, the slightly positive correlation with normal sperm morphology seems to be applicable only in cases of oligozoospermia. Significant negative correlation of the number of peroxidase‐positive cells were found for both maximal inducible acrosome reaction (Pu2003= 0.0219) and the inducibility of acrosome reaction (Pu2003=u20030.0370), indicating a rather deleterious effect of leucocytes on this important sperm function. Concerning the result in the in vitro fertilization programme, none of the examined parameters (PMN elastase, concentration of round cells and peroxidase‐positive cells) showed a correlation with either fertilization or pregnancy. This result seems to be reasonable as severely damaged spermatozoa and leucocytes are eliminated from the ejaculate by different sperm separation methods. Interestingly, a significant negative correlation of the TUNEL assay as a measure of sperm DNA fragmentation was found only with pregnancy (Pu2003=u20030.006) but not with fertilization. As DNA fragmentation can also be caused by ROS that are generated by leucocytes, this causality should not be neglected.

Sperm morphology and male urogenital infections.

Summary. The aim of this study was to investigate the influence of urogenital infections as indicated by leukocytospermia on human sperm morphology, diagnosed cytologically and by means of a leukocyte peroxidase test. A basic semen analysis, including a leukocyte peroxidase test, was prospectively performed on 150 consecutive semen samples. Cytology smears were microscopically investigated for the presence of WBC and the results expressed on a 4 point scale as ± to + + + WBCs/high power field (HPF). ROC curve analysis indicated that for cases with more than ± WBC/HPF the peroxidase determined WBC count cut‐off value was >0.25 times 106 WBC ml−1 with a sensitivity of 75% and specificity of 90%. The presence of more than ±WBC/HPF was negatively correlated with sperm morphology characteristics studied. The mean (±SD) percentage of morphological normal spermatozoa was 7.0% (SD 4.4) in the WBC negative group (n=134) compared to 4.3% (SD 3.5) in the WBC positive (n=16) group (P<0.0001). There was also an associated increase, 15.3% (SD 13.3) to 23.6% (SD 13.8), in the percentage of spermatozoa with elongated head forms in the WBC positive group (P=0.0218). No other effect on sperm and acrosome morphology could be found. With the peroxidase determinations there was also a tendency in the WBC positive group (n=10) towards poorer sperm morphology characteristics, but these changes were not statistically significant. The presence of urogenital infections as diagnosed cytologically was associated with statistically poorer sperm morphology characteristics. This statistical relationship was not found in the peroxidase diagnosed leukocytospermia positive groups.

Intracytoplasmic sperm injection with testicular spermatozoa in men with azoospermia

AbstractPurpose: The aim of the study was to gain an insight into the optimal management of the infertile couple with the husband suffering from azoospermia.nMethods: One hundred and forty-two intracytoplasmic sperm injection (ICSI) cycles performed with testicular extracted spermatozoa were retrospectively analysed. The following factors were investigated for their possible influence on fertilization, cleavage, damage, pregnancy, and ongoing pregnancy rates; the use of fresh, cryopreserved, and preincubated (24 h) spermatozoa and the etiology of the husbands azoospermia (obstructive and nonobstructive). All microinjections were performed with apparently normal spermatozoa—a head with a tail of normal length. In 116 cycles at least two embryos were available for transfer.nResults: The overall fertilization, clinical pregnancy, and ongoing pregnancy rates obtained for the 116 cycles were 65.0, 30.2, and 22.4% respectively. Similar outcomes were obtained for cycles using fresh testicular and cryopreserved testicular spermatozoa. Similarly, no significant differences were obtained between the cycles using spermatozoa from obstructive or nonobstructive azoospermic patients. An increase in motility after a 24-h preincubation was observed, and although this group was relatively small (n = 17), a significant improvement in fertilization (73.7%) and pregnancy (53.9%) rate was obtained when the testicular sample was preincubated for 24 h. This improvement prevailed in the obstructive azoospermic group, but was less pronounced in nonobstructive patients.nConclusions: This study shows that the outcome of fresh and frozen–thawed testicular spermatozoa in ICSI is comparable, obstructive and nonobstructive etiologies perform the same, and that preincubation of testicular spermatozoa results in increased fertilization and pregnancy rates. All testicular biopsies are therefore performed the day before oocyte retrieval, superfluous spermatozoa cryopreserved, and the remaining testicular homogenate preincubated for the 24 h prior to oocyte retrieval. With this regime, most azoospermic patients are treated successfully, irrespective of the use of fresh or frozen–thawed spermatozoa from obstructive or nonobstructive cases.

Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution

Summary. The hemizona assay (HZA) was developed to evaluate sperm binding potential using microbisected human zona pellucida. In this study, eight human oocytes stored in a buffered salt solution for 60 days were bisected into two identical hemispheres (hemizonae) and coincubated with the spermatozoa from a fertile man. All evaluated spermatozoa were tightly bound to the outer surface or had begun penetration into the zona pellucida. The hemizonae with bound spermatozoa were prepared and fixed for transmission electron microscopy (TEM) using standard techniques. Among the 108 sperm bound to the zone we were able to evaluate 25 by TEM. Twenty (80%) of the zona bound spermatozoa were partially or completely acrosome reacted, while six (20%) of the zona bound sperm had intact acrosomes. Acrosome intact, partially acrosome reacted and completely reacted spermatozoa were observed within the zona. Penetration pathways or tunnels were seen within the zona matrix. The results illustrate, that typically spermatozoa tightly bound the human zona pellucida show induction of the acrosome reaction. Importantly, following storage of human eggs in salt solution (buffered to 7.4), the zona pellucida retain their biological and functional characteristics for at least 90 days.

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.

Sperm-oocyte interaction: studies on the kinetics of zona pellucida binding and acrosome reaction of human spermatozoa

Summary. Successful sperm‐oocyte interaction depends, among other things, on sperm capacitation, which is defined by acrosomal and motility alterations. In the study described here the authors evaluated different aspects of this gamete interaction in humans. Specifically, the authors studied (1) the relationship between the number of spermatozoa bound to the zona pellucida and sperm concentration and incubation period, (2) the capacitation status and kinetics of acrosome reaction among the zona‐bound spermatozoa, and (3) the effect of human follicular fluid on the zona‐binding potential and acrosome status of spermatozoa from different men. The results indicated a concentration of 107 cells ml−1 after 15 min of coincubation to be the optimum for zona binding. The number of sperm bound after 0, 3 and 5 h of incubation was the same. In addition, spermatozoa incubated for 3 or 5 h underwent the acrosome reaction (range 9–43%) on the zona surface within 15 min of binding. The maximum percentage of acrosome‐reacted spermatozoa was reached after 60 min of binding. Follicular fluid affected the sperm populations selectively, since it did not influence zona binding capacity in all cases. The data enhances the authors understanding of critical events occurring before fertilization.

A novel approach for the selection of human sperm using annexin V-binding and flow cytometry

OBJECTIVEnTo develop a method whereby sperm with phosphatidylserine externalization can be separated from those without this feature. Because annexin V binds phosphatidylserine, this study is using this feature to select functional spermatozoa. In addition, the relationship between annexin V binding in human spermatozoa and normal sperm morphology according to strict criteria was to be assessed.nnnDESIGNnProspective study.nnnSETTINGnDepartment of Obstetrics and Gynaecology of Stellenbosch University at Tygerberg Academic Hospital, Tygerberg, South Africa.nnnPATIENT(S)nSemen from 14 healthy sperm donors. Exclusion criterion was the presence of less than 20 x 10(6)/mL total motile spermatozoa in the original sample.nnnMAIN OUTCOME MEASURE(S)nAnnexin V-negative sperm, annexin V-positive sperm, normal sperm morphology.nnnINTERVENTION(S)nAn aliquot of a semen sample after double density gradient centrifugation was incubated with annexin V fluorescein isothiocyanate conjugate (FITC). Cell fluorescence signals were determined using a FACScalibur flow cytometer equipped with a FACSSort fluidic sorting module. The sorting procedure delivered two sperm subpopulations: annexin V-negative and annexin V-positive. Morphology slides were made and the sperm morphology was assessed according to strict criteria.nnnRESULT(S)nThere was a significant enrichment of annexin V-negative sperm as well as morphologically normal sperm in the annexin V-negative subgroup after separation with flow cytometry. The percentage of morphologically normal sperm increased from 8.3% in the control to 11.9% in the annexin V-negative fraction, whereas the percentage of annexin V-positive sperm decreased to 5.7%.nnnCONCLUSION(S)nThe annexin V-negative sperm subpopulation had morphologically superior quality sperm compared to annexin V-positive sperm. It is important to select morphologically normal sperm during intracytoplasmic sperm injection (ICSI) as it may contribute to increased implantation and pregnancy rates (PR).

Effects of different staining and washing procedures on the results of human sperm morphology evaluation by manual and computerised methods

Summary. The purpose of this study was to investigate the effect of different staining and washing procedures on the results of human sperm morphology evaluation by manual and computerised methods. Furthermore, it was intended to find the staining and washing combination which would provide optimal readability for computer‐assisted sperm morphology evaluations. In phase one, four staining methods were evaluated for smears prepared from the resulting samples following a two times washing procedure. In phase two, 20 semen samples were used to compare the Diff‐Quik and Papanicolaou staining methods, following one and two washes. All manual readings, of Papanicolaou and Diff‐Quik stained smears, were comparable with each other, with means between 7.3% and 7.9% normal spermatozoa. All the manual readings were also comparable to the computer readings of the Diff‐Quik slides following one and two washes with means of 9.0% and 5.9%, respectively. However, due to the higher computer readings found for the Papanicolaou stained smears, with means of 13.9% and 13.5% following one and two washes, respectively, a statistically significantly difference between overall computer and manual readings was found (Wilks Lamda, P = 0.0002). Taking all data into consideration, it could be concluded that the one wash Diff‐Quik stained smears was the optimal preparation method for computerised sperm morphology evaluation, comparing favourably with manual evaluations.

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.

Is metformin indicated as primary ovulation induction agent in women with PCOS? A systematic review and meta-analysis

Background: A recent meta-analysis has proven that metformin (M) is highly effective for ovulation induction in the clomiphene citrate (CC)-resistant patient. There is uncertainty whether M should be introduced as a primary ovulation induction agent in polycystic ovarian syndrome (PCOS). Methods: We conducted a systematic review and meta-analysis to establish if M is better when given alone or in combination with CC (CC+M) when compared with CC alone. This systematic review studied live birth delivery rate as the primary outcome. Results: We identified 14 prospective trials. Analysis of these results showed a reduction in the live birth rate in the group of patients treated only with M when compared with CC alone (OR = 0.48, 95% CI 0.31–0.73, p = 0.0006). An increase in ovulation (OR = 1.6, 95% CI 1.2–2.1, p = 0.0009) and pregnancy rate (OR = 1.3, 95% CI 1.0–1.6, p = 0.05) with CC+M when compared with CC alone was reported, but no difference was found when live birth rate was analyzed (OR = 1.1, 95% CI 0.8–1.5, p = 0.61). Conclusion: CC alone is superior to M alone regarding live birth rate and ovulation. The combination (CC+M) is superior to CC alone as a primary method for ovulation induction and to achieve pregnancy in PCOS. However, when addressing live birth rate, no statistically significant difference could be demonstrated. Because of the side effects profile and contraindications of M, we believe M should not be indicated as a primary ovulation induction agent in women with PCOS.