Roelof Menkveld

Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial

OBJECTIVE To study the effects of folic acid and zinc sulfate treatment on semen variables in fertile and subfertile men. DESIGN Double-blind, placebo-controlled interventional study. SETTING Two outpatient fertility clinics and nine midwifery practices in The Netherlands. PARTICIPANT(S) One hundred eight fertile and 103 subfertile men. INTERVENTION(S) Both groups were randomly assigned to receive one of four treatments for 26 weeks: folic acid and placebo, zinc sulfate and placebo, zinc sulfate and folic acid, and two placebos. Folic acid was given at a daily dose of 5 mg, and zinc sulfate was given at a daily dose of 66 mg. MAIN OUTCOME MEASURE(S) Before and after treatment, standardized semen and blood samples were obtained for determinations of sperm concentration, motility, and morphology according to World Health Organization guidelines; semen morphology according to strict criteria; and blood folate and zinc concentrations. Effects of the four interventions were evaluated separately in subfertile and fertile men. RESULT(S) Subfertile men demonstrated a significant 74% increase in total normal sperm count and a minor increase of 4% abnormal spermatozoa. A similar trend was observed in fertile men. Pre-intervention concentrations of folate and zinc in blood and seminal plasma did not significantly differ between fertile and subfertile men. CONCLUSION(S) Total normal sperm count increases after combined zinc sulfate and folic acid treatment in both subfertile and fertile men. Although the beneficial effect on fertility remains to be established, this finding opens avenues of future fertility research and treatment and may affect public health.

DNA fragmentation of spermatozoa and assisted reproduction technology

Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in the ejaculate and in the spermatozoa themselves. These data were correlated with fertilization and pregnancy data from IVF and intracytoplasmic sperm injection (ICSI) patients. Sperm DNA fragmentation did not correlate with fertilization rate, but there was a significantly reduced pregnancy rate in IVF patients inseminated with TUNEL-positive spermatozoa. ICSI patients exhibited the same tendency. This implies that spermatozoa with damaged DNA are able to fertilize an oocyte, but at the time the paternal genome is switched on, further development stops. The determination of ROS in the ejaculate and the percentage of ROS-producing spermatozoa revealed markedly stronger correlations between sperm functions (i.e. motility) and the percentage of ROS-producing spermatozoa. The influence of seminal leukocytes, known to produce large amounts of oxidants, on sperm DNA fragmentation should not be neglected.

CHROMOSOME STUDIES IN 496 INFERTILE MALES WITH A SPERM COUNT BELOW 10 MILLION/ML

SummaryChromosome studies were performed on 106 men with azoospermia and 390 men with oligozoospermia (consistant sperm count below 10 million/ml). Constitutional chromosome abnormalities were found in 14.1% of the azoospermia group and in 5.1% of the oligozoospermia group. An overall incidence of 7.1% constitutional abnormalities indicates that this criterion of selection may be advisable for routine chromosome analysis of infertile men. A reduction of 25% in the workload increases the yield of chromosome abnormalities in the group of infertile men to 10–14 times above that expected in the normal population.

Sperm morphology: assessing the agreement between the manual method (strict criteria) and the sperm morphology analyzer IVOS * †

OBJECTIVES To correlate the percentage normal morphology reported by different observers and a computerized method (IVOS; Hamilton-Thorne Research, Beverly, MA) on a slide-by-slide basis using strict criteria: [1] Experienced observer (T.F.K.) versus experienced observer (R.M.), [2] experienced observer (T.F.K.) versus sperm morphology analyzer (IVOS), and [3] repeatability of normal and abnormal cells (IVOS). DESIGN SETTING, PATIENTS: Slides from 30 different patients from the Tygerberg IVF program were selected randomly. Microscopic fields and sperm cells were chosen randomly and percent normal morphology was recorded (objectives 1 and 2). The same slides were used and a cell-by-cell repeatability was done as outlined (objective 3). RESULTS Experiment 1 (objective 1): there was no significant bias between T.F.K. and R.M. The limits of agreement were 8.6% and -7.3%. The SDs were not significantly different (P = 0.1283). The Spearman correlation coefficient between readers was 0.83. Experiment 2 (objective 2): the same findings were reported but the limits of agreement were 12.1% and -15.5%. The Spearman correlation coefficient was 0.85. The limits of agreement was tighter below 20% normal forms (+8.4 and -6.6). Experiment 3 (objective 3) (repeatability): 255 cells were analyzed three times in succession. Estimating pairwise agreement, the kappa statistic for the pairs are 0.85, 0.80, and 0.85, respectively, which compares favorably with the second canonical moment of 0.8329 (kappa = 0.83). DISCUSSION The computers ability to classify normal morphology per slide is promising. Below 20% normal forms, the limit of agreement is tight. Because of the 6% higher reading compared with the manual method, different thresholds possibly will be developed to identify subfertile from fertile patients. The computer gives excellent repeatability of normal and abnormal cells. Based on results obtained, this system can be of clinical value both in IVF units and andrology laboratories but more clinical data is required in this field.

Morphometric dimensions of the human sperm head depend on the staining method used

BACKGROUND Assessment of sperm morphology (including morphometry) is extensively used to determine one of the qualities of a semen sample and depends on the differential staining of spermatozoa. A staining technique should cause as little change to sperm dimensions and form as possible in order to reliably evaluate the morphometric features of the sperm. Various staining techniques have been employed, but only a few have been recommended by the World Health Organization and are amenable to automated sperm morphometry analysis. Our study was aimed at comparing the effect of three staining techniques [Papanicolaou (PAP), Rapidiff (RD) and SpermBlue (SB)] on human sperm head dimensions and to compare these with the head dimensions in fresh semen. METHODS Smears made from human semen samples (n = 24) were stained according to the three staining techniques and sperm head morphometry was assessed with the Sperm Class Analyzer. Head dimensions of fresh spermatozoa were measured with a digital calliper on a computer screen. The minimum number of spermatozoa to be analyzed to represent the sperm population and the degree of inter-laboratory variation were determined. Electron micrographs from the same semen samples were used to determine the actual acrosome coverage of the spermatozoa in the semen (n = 7) in order to verify the results of the automatic analyses. RESULTS The osmolality of human semen differs from that of the RD and PAP fixatives and stains, but is more similar to the SB fixative and stain. At least 100 spermatozoa should be analyzed to include a representative sample of the sperm population. RD caused sperm heads to swell, PAP caused them to shrink and SB had no significant effect on sperm head dimensions when compared with spermatozoa in fresh semen. Very little inter-laboratory variations were found. The percentage acrosome coverage was significantly different between the three staining techniques, as well as between the RD and PAP stains and the manual measurements obtained using the electron micrographs. CONCLUSIONS Different staining techniques change the morphometric dimensions of the human sperm head, probably due to the fact that either the fixatives or stains are not iso-osmotic in relation to human semen. Since these changes in sperm head dimensions are not uniform, care should be taken when selecting a staining technique. Ideally, stained spermatozoa should have dimensions as close to spermatozoa in fresh semen as possible, as was found with the SB staining method, resulting in accurate evaluations of sperm head morphometry.

Measurement and significance of sperm morphology

The measurement or evaluation and clinical significance of human sperm morphology has always been and still is a controversial aspect of the semen analysis for the determination of a males fertility potential. In this review the background of the development of the evaluation criteria for sperm morphology will be discussed. Aspects of criticism on the strict criteria definition and use of the criteria for sperm morphology evaluation will be discussed as well as possible reasons for the decline in normal sperm morphology values and how we can compromise for this phenomenon resulting in the very low normal reference value as published in the 2010 WHO manual for the Examination and Processing of Human Semen. One of the possible solutions may be to give more attention to a limited number of abnormal sperm morphology categories and the inclusion of sperm morphology patterns. It is concluded in this review that if done correctly and with care and with strict application of existing guidelines as outlined in the 2010 WHO manual, sperm morphology measurement still has a very important role to play in the clinical evaluation of male fertility potential.

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 106 leucocytes ml−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 (P < 0.0001). However, a negative correlation of peroxidase‐positive cells with the sperm concentration was only found in oligozoospermic patients (P < 0.0001). 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 (P = 0.0219) and the inducibility of acrosome reaction (P = 0.0370), 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 (P = 0.006) 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.

Obesity is associated with increased seminal insulin and leptin alongside reduced fertility parameters in a controlled male cohort.

BackgroundObesity appears to be associated with male reproductive dysfunction and infertility, although this has been inconsistent and inconclusive. Insulin and leptin are known mediators and modulators of the hypothalamus-pituitary-testes axis, contributing to the regulation of male reproductive potential and overall wellbeing. These hormones are also present in semen influencing sperm functions. Although abdominal obesity is closely associated with insulin resistance (hyperinsulinaemia), hyperleptinaemia and glucose dysfunction, changes in seminal plasma concentrations of insulin, leptin and glucose in obese males has not previously been investigated.MethodsThis small case controlled study assessed serum and seminal concentrations of insulin, leptin and glucose in obese (BMI > =30; n = 23) and non-obese (BMI < 30; n = 19) males. Following a detailed medical history and examination, participants meeting the inclusion criteria were entered for data analysis. Body parameters such as BMI, waist and hip circumference and the waist hip ratio were measured. Serum and semen samples were collected and assayed for insulin, leptin and glucose. Semen samples also underwent a standard semen analysis, with sperm mitochondrial membrane potential (MMP) and DNA fragmentation (DF).ResultsObesity was associated with increased serum and seminal insulin and leptin, with no significant difference in seminal glucose. Serum and seminal concentrations of insulin and leptin were positively correlated. Furthermore, obesity was associated with decreased sperm concentration, sperm vitality and increased MMP and DF, with a non-significant impact on motility and morphology.ConclusionsHyperinsulinaemia and hyperleptinaemia are associated with increased seminal insulin and leptin concentrations, which may negatively impact male reproductive function in obesity. Insulin was also found to be highly concentrated in the seminal plasma of both groups. This data will contribute to the contradictive information available in the literature on the impact of obesity and male reproduction.

Relationship between zona pellucida-induced acrosome reaction, sperm morphology, sperm-zona pellucida binding, and in vitro fertilization

OBJECTIVE To evaluate the possible relationships between sperm morphology, acrosome responsiveness to solubilized human zona pellucida, and sperm-zona binding potential among [1] consecutive andrology referrals and [2] randomly selected in vitro fertilization (IVF) cases. DESIGN Prospective analytical study. SETTING Academic training hospital.Randomly selected couples consulting for infertility. INTERVENTION(S) Acrosome reaction response to solubilized human zona pellucida was recorded. MAIN OUTCOME MEASURE(S) We determined the difference in the percentage of sperm that acrosome reacted after exposure to solubilized zona pellucida and spontaneous acrosome reaction. The results were expressed as percentage zona induced acrosome reaction (ZIAR). RESULT(S) Data were analyzed using correlation coefficients (r) and receiver operator characteristics (ROC curve analyses). The ROC curve analyses indicated ZIAR to be a sensitive indicator for fertilization failure during IVF therapy, with sensitivity and specificity of 81% and 75%, respectively. For andrology referrals, a positive and statistically significant correlation existed between ZIAR data and sperm morphology (r = 0.65) and sperm-zona binding (r = 0.57). CONCLUSION(S) ZIAR results provide further information regarding dysfunctional sperm and can be used as an additional diagnostic test. Our results predicted fertilization failure during IVF treatment.