Office-based sperm concentration: A simplified method for intrauterine insemination therapy.

BACKGROUND: Intrauterine insemination (IUI) could become preferred to more invasive and expensive techniques of assisted reproduction therapy (ART) and should be offered as the first choice in cases with no female factors and mild male factor subfertility. However, developing countries and especially their rural areas often lack the necessary equipment and laboratory facilities. OBJECTIVE: To describe a simplified one-step method to determine the sperm concentration range for IUI therapy. METHODS: Semen samples from 51 sperm donors were used. Following swim-up separation, the sperm concentration of the retrieved motile fraction was counted, as well as progressive motile sperm using a standardised wet preparation. The number of sperm in a 10 µL droplet covered with a 22 x 22 mm coverslip was counted under 400 x total magnification. The observed numbers of retrieved motile sperm were divided into three groups: < 40, 40-100 and > 101 spermatozoa as recorded per intial estimation on the wet preparation. RESULTS: The mean (standard deviation) estimated sperm concentration for each group compared with actual counts per Neubauer counting chamber were: estimated < 40 sperm (n = 14), mean 20 (8), Neubauer count 2.5 x 10(6)/mL; estimated 40-100 sperm (n = 14), mean 71 (15), Neubauer count 16 x 10(6)/mL; and estimated > 100 sperm (n = 23), Neubauer count 48.3 (21.7) x 10(6)/mL. CONCLUSION: The results with IUI in male subfertility cases reported by Ombelet et al. in 1995 support the concept of first-line treatment of infertility by three to four cycles of IUI therapy in selected cases.

Semen analysis workshops: 17 years' experience.

The study reports on the results recorded during a series of semenology workshops presented from 1997 to 2014. The results were obtained from training workshops that were conducted in 6 continents in 38 cities including 1124 individuals. All workshops consisted of 2 sessions namely a pre-and post-training session. Results recorded from the pre-training sessions were used as a baseline value of current knowledge. In most cases pooled fresh or cryopreserved semen samples were provided by the organizing institution. The mixed effect linear regression model showed a significant decrease in the mean scores from the pre-training scores, p < .001. Training of technicians as well as regular proficiency testing will ensure continuous communication with the referring laboratory.

Sperm morphology training in India.

Sperm morphology is regarded as possibly the most consistent sperm variable that is related to in vitro and in vivo fertilisation success or failure. There is also a re-emerging awareness of andrology, particularly in developing countries where healthcare resources are often limited. This study aimed to record the importance of hands-on training on the sperm morphology reading skills among 174 Indian infertility workers. During a series of five workshops presented by the Indian Academy of Clinical Embryologists, delegates were requested to record the percentage normal spermatozoa on pre-stained Diff Quick slides. A total of four slides each containing different percentages normal spermatozoa were used during the workshops. The delegates were requested to record the percentage normal spermatozoa prior and after training. The results were expressed as the percentage difference between the reference value and the pre-training and post-training values reported by the workshop delegates. The percentage difference indicated a significant improvement in the reading skills, for example from 253 ± 244% to 20 ± 93% for slide 1. Similar values were recorded for slide 2, 3 and 4. If we consider the present findings as representative of medical laboratory scientist's sperm morphology reading skills, clinicians should be concerned about the diagnostic quality as far as male infertility is concerned.

Relationship between human spermatozoa-hyaluronan-binding assay, conventional semen parameters and fertilisation rates in intracytoplasmic spermatozoa injection.

Selecting the best spermatozoa for intracytoplasmic spermatozoa injection (ICSI) has recently been a topic of great interest among embryologists. The study aimed to evaluate the relationship between the spermatozoa-hyaluronan-binding assay (HBA), routine semen analysis results and fertilisation rates as recorded during conventional ICSI therapy. Ninety-one patients undergoing conventional ICSI treatment in the Medfem Fertility Clinic in Johannesburg (South Africa) were included in the study. A total of 797 oocytes were injected of which 457 oocytes fertilised (57.3%, range 0-100%). None of the semen parameters correlated with the fertilisation rates (Table 2). HBA scores, however, revealed a highly significant association (p ≤ 0.0001) with the fertilisation rates. The HBA scores also correlated significantly with the biochemical pregnancy values (Spearman r = 0.24, P = 0.02, 95% CI 0.039-0.43); however, the HBA scores did not correlate with the clinical pregnancy rates (Spearman r = 0.14, P = 0.16, 95% CI -0.06 to 0.34). No correlation was recorded between HBA and the standard semen parameters. The study showed that HBA is significantly associated with fertilisation in conventional ICSI. The HBA scores were also significantly associated with the fertilisation rates and biochemical pregnancies.

How accurate is sperm morphology as an indicator of sperm function?

Sperm morphology has been consistently correlated with fertilisation success or failure. The clinical relevance of the percentage normal spermatozoa has been a widely discussed topic amongst infertility specialists and scientists. This study aimed to evaluate the role of sperm morphology as an indicator of additional sperm functions among 114 andrology referrals. The sperm functions that were investigated included chromatin packaging quality (CMA3 test (n = 109), zona-induced acrosome reaction (ZIAR test; n = 36), hemizona assay (HZI; n = 36) and progressive motility (n = 47). Chromatin packaging quality had a negative and significant (P = 0.0001, r = -0.74) correlation with the percentage normal spermatozoa, while progressive motility had a significant and positive correlation (P = 0.0001, 0.59). Accurate sperm morphology scoring as described by the WHO 2010 manual can therefore be used as an indicator of specific sperm functions.

Semen analysis workshops in India and Africa: the vital role of training and external quality control programmes.

Two hundred and fifty seven individuals from 16 African and Indian andrology laboratories attended several -semenology workshops from 1997 to 2013. The external quality control programme in Africa indicated the ability of participants to maintain the technology skills following a hands-on training. The pre-training sessions of the workshops indicated a total lack of knowledge how to perform a semen analysis according to the WHO manual. This was the case for morphology, motility and concentration. The results of this report underline the vital role of training as well as external quality control programmes.

Sequential analysis of sperm functional aspects involved in fertilisation: a pilot study.

The development of diagnostic techniques in andrology as a second level of approach to the diagnosis of male factor infertility has enthused the focus of researchers on the development of a sequential diagnostic programme for these men. Semen samples of 78 men form couples undergoing in vitro fertilisation therapy were used in the study. The semen samples were used to test sperm functional aspects known to interfere with fertilisation. These tests included semen profile, DNA integrity, apoptosis, chromatin packaging, acridin orange staining, zona binding capacity, zona-induced acrosome reaction (AR). Results were correlated with fertilisation outcome. Statistical analyses of the recorded data were carried out using a logistic regression analysis model on all sperm functional tests. A negative and significant association with the fertilisation rates was recorded for DNA damage (r = -0.56; P ≤ 0.0005). A positive significant correlation was recorded between fertilisation rates and sperm with normal DNA (r = -0.57, P ≤ 0.0004), and zona-induced AR (r = 0.33, P ≤ 0.002). Diagnostic andrology can be regarded as a mandatory part of the male factor patient's work-up schedule to assist clinicians with the most suitable therapeutic modality to follow.

Accurate sperm morphology assessment predicts sperm function.

Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.

Temperature controlled centrifugation improves sperm retrieval.

Sperm retrieval techniques form an integral part of the assisted reproductive programme. The success of sperm separation is measured by the number of motile sperm retrieved from a given semen sample. The study aimed to evaluate the effect of temperature during sperm preparation events on the number and percentage motile sperm retrieved following a double wash swim-up procedure. Thirty semen samples were obtained from 10 normozoospermic donors. After collection samples were divided into two aliquots, one aliquot was placed in an incubator at 34 °C, while the second aliquot was left at room temperature (25 °C). Sperm motility assessments were recorded with a computer assisted sperm analyser. Motile sperm fractions were retrieved from the semen samples following a double wash swim-up technique. Two tubes were prepared for each experiment. Tubes were placed in two different centrifuges: (i) SpermFuge (Shivani Industries, India) with temperature centrifuge control (34 °C) and (ii) Sigma with no temperature control facilities. Both centrifuges were set at 484 g for 5 min. Following the second wash, sperm pellets were layered with culture medium, and sperm was allowed to swim up. Supernatants were removed and analysed for sperm concentration and motility values. Percentage motile sperm was transformed to ARCSIN values and results of the two centrifugation methods at 34 °C and room temperature were compared with Mann-Whitney test for independent samples. The mean sperm concentration retrieved at 34 °C was 43.8 ± 50 (SpermFuge) and 32.7 ± 21 (Sigma) (P < 0.05), compared to retrieved concentration at room temperature namely, 30.9 ± 33 (SpermFuge) and 30.6 ± 17 (Sigma) (P ≥ 0.05). The mean percentage motile sperm at 34 °C was 64.0 ± 19 (SpermFuge) and 44.2 ± 24 (Sigma) (P = 0.02), while at room temperature the percentage motile sperm was 54.7 ± 17 (SpermFuge) compared to 46.5 ± 14 (Sigma) (P ≥ 0.05). Centrifuge temperature and incubation temperature significantly influenced the percentage retrieved motile sperm. The use of temperature-controlled sperm preparation might have clinical value for men with poor sperm motility values.

The effect of pulsed 900-MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa.

Several recent studies have indicated that radiofrequency electromagnetic fields (RF-EMF) have an adverse effect on human sperm quality, which could translate into an effect on fertilization potential. This study evaluated the effect of RF-EMF on sperm-specific characteristics to assess the fertilizing competence of sperm. Highly motile human spermatozoa were exposed for 1 h to 900-MHz mobile phone radiation at a specific absorption rate of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed using computer-assisted sperm analysis. Significant reduction in sperm head area (9.2 ± 0.7 µm² vs. 18.8 ± 1.4 µm²) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) was reported among exposed sperm compared with unexposed controls. Sperm-zona binding was assessed directly after exposure using the hemizona assay. The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p < 0.05), respectively. This study concludes that although RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition, a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilization potential.

Comparison between propidium iodide and 7-amino-actinomycin-D for viability assessment during flow cytometric analyses of the human sperm acrosome.

Evaluation of the acrosome reaction can shed light on the fertilising competence of spermatozoa. To eliminate false-positive results when evaluating the acrosome status of human sperm cells, two viability probes propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) were compared for their ability to stain nonviable cells post-fixation and permeabilisation. Both the mean fluorescence and % dead cells differed significantly with time (P < 0.0001). Unlike PI, 7-AAD did not leach from cells and fluorescence remained stable for up to 4 h. Furthermore, 7-AAD proved to be a proficient marker to exclude dead sperm cells during flow cytometric evaluation of ionophore-induced acrosome reaction.

Can a cumulus cell complex be used to select spermatozoa for assisted reproduction?

Since the onset of intracytoplasmic sperm injection, researchers have intensified the search for the ideal spermatozoa to be used for injection. The aim of this study was to record the functional role of cumulus cell interaction with human spermatozoa as far as capacitation, acrosome reaction, morphology, zona binding and chromatin packaging quality are concerned. Using a previously described cumulus oophorus model, we recorded specific sperm functional aspects of sperm populations that transverse a cumulus cells mass. Control spermatozoa were kept under similar experimental conditions in the culture media only. Results indicated cumulus cells to be beneficial to spermatozoa as far as functional and capacitational events are concerned. The mean percentage of morphologically normal spermatozoa in the control sample was 6.9%, while the spermatozoa that traversed the cumulus oophorus (test) had a significantly higher percentage of normal forms (mean 9.5%; P < or = 0.01). We observed a decline in the percentage of CMA3-positive spermatozoa when we compared the control population (49.1%) to the test, i.e. 38.4%, (P = <0.05), thus implying that the spermatozoa with good chromatin condensation increased during cumulus penetration. Significantly more (P < or = 0.01) acrosome-reacted spermatozoa were found in the penetrated spermatozoa (mean 23%) than in the control spermatozoa (mean 11%). The test spermatozoa had a higher zona binding capacity with significantly more (P < or = 0.01) tightly bound spermatozoa on the hemizona (61 +/- 15) than the control spermatozoa (47 +/- 18). In the absence of sophisticated and expensive sperm selection products, the use of a cumulus model to select spermatozoa for intracellular sperm injection seems to be an alternative method.

Morphology training is compulsory to ensure relevant clinical results.

The study aimed to evaluate the quality of sperm morphology reading skills of andrology technologists in a developing country who are responsible for the analysis of semen in their institutions or clinics. The 1-day workshop was held as a post-congress training course during the second World Congress on Practical Infertility Management and Human Reproduction, Mumbai, India. The workshop format consisted of four sections namely (i) pre-training test, (ii) lectures on morphometric characteristics and details of normal and abnormal spermatozoa (iii) a consensus training session and (iv) a hands-on post-training test. The results revealed that the workshop participants had very little or no knowledge of sperm morphology, because the pre-training % relative difference between the reference value and the participants was 188%. Following training, however, the % difference decreased to 37% and minus 22% post-training tests. These somewhat alarming results highlight the need for training opportunities and facilities for andrology technologists.

Does training assist medical laboratory scientists with better evaluation of sperm morphology?

This study aimed to evaluate the quality of sperm morphology evaluation skills of 16 technologists who are responsible for semen analyses in their institution. The 1-day workshop was held at the Centre for Research in Reproductive Health in Sagamu, Nigeria. Participants were requested to complete a questionnaire that provided information regarding their experience and training as technologists in their institution. The workshop consisted of 4 sections, namely (i) pre-training test, (ii) lectures on morphometric characteristics and details of normal and abnormal sperm (iii) a laboratory-based hands-on, post training test and (iv) a consensus training session. The findings of the questionnaire indicated that none of the participants had any previous formal training and that all participants had very little knowledge about the morphological appearance of normal spermatozoa. These findings were supported by the results obtained during the pre-training session that showed a mean percentage difference of 24% between the reference and participant's values. These somewhat alarming results highlight the need for training facilities for technologists who work in the clinical diagnostic field of reproductive science.

Lessons learned from a sperm morphology quality control programme.

Although sperm morphology is the sperm parameter that is consistently correlated with in vitro and in vivo fertilisation, its value is often challenged due to discrepancies in reports. As sperm morphology is subjectively evaluated, the outcome of the report is entirely based on the experience and technical skill of the laboratory technician responsible for semen analyses. The study aimed to record the value of quarterly monitoring and refresher courses on morphology reading skills of technicians over a period of 40 months. Nineteen individuals from 12 different andrology laboratories were enrolled to a sperm morphology quality control programme after initial training sessions. Five of the 19 participants attended annual refresher courses. Two limits of error, namely +/- 0.5 SD and +/- 0.2 SD scores, were used to record technician deviation from the reference laboratory. Fifteen of the 19 individuals (78%) consistently reported sperm morphology readings that were within the +/- 0.5 SD limits of error. Furthermore, five of the participants, who attended annual refresher training courses, consistently reported results that were within the +/- 0.2 SD limits of error. For the first time, it has been illustrated that excellent sperm morphology reading skills can be achieved and maintained by initial training sessions, followed up with a continuous external quality control programme and annual refresher courses.

The role of paternal chromosomes and sperm morphology on the outcome of intracytoplasmic sperm injection.

The study investigated the possible relationship between the X/Y chromosomes bearing spermatozoa and the outcome of intracytoplasmic sperm injection (ICSI) therapy and morphological appearance/shape (Tygerberg criteria) of the sperm cell injected into the oocyte during ICSI therapy and fertilisation. Thirty-nine patients were recruited for the study from an assisted reproductive programme. Semen samples were prepared by using gradient centrifugation techniques. Prior to injection sperm images were captured using high-quality video graphic equipment. Sperm selection was based on the concept of 'best-looking' spermatozoa i.e. spermatozoa lacking gross and obvious malformations such as broken necks, cytoplasmic droplets, amorphous or elongated heads. Photomicrographs of each sperm cell were produced from video footage. The photographical material was used to determine the basic shape and the actual length-to-width ratio of the injected sperm heads. Embryo biopsies and fluorescent in situ hybridisation (FISH) was performed on 12 randomly selected couples from a set of 39. Embryos were evaluated on day 3 for development and embryo transfer. Embryo biopsies and FISH analyses were performed on those embryos that showed no developmental potential following injection. It was found that 70% of the embryos that showed no developmental potential were Y chromosome-bearing embryos. The sperm selection process for ICSI based on the approach of choosing the 'best-looking' spermatozoon in the ejaculate seems to provide cells that can be classified as normal based on the length-to-width ratio set by the World Health Organization for normal cells.

The establishment of sperm morphology satellite training laboratories in Africa.

Summary Discrepancies in the results of semen analysis can be caused by a variety of factors, for example, a microscope for semen analysis can be situated in the practice of an urologist, cytologist or microbiologist, or even in a health care office in the countryside in a developing country. The study aimed to evaluate the (i) sperm morphology assessment skills of the participants who attended WHO seminology workshops and (ii) to appraise the possibility of using trained technologists to set up satellite training facilities in their own area. The Centre for Research in Reproductive Health, Ogun State University Teaching Hospital (Nigeria) initiated training sessions in their area. Subsequently, 16 technologists were trained in three sessions. Papanicolaou-stained slides, supplied by Tygerberg were used as test material. Pre- and post-training results were used to calculate the mean percentage of normal cells as well as the percentage deviation from the reference laboratory. The mean percentage normal spermatozoa recorded by the Nigerian trainees during pre-training evaluations differed by 38% from the reference value, compared with a post-training difference of 16% (P < 0.001). Tertiary academic hospitals can play an important educational role in the region. The present results underline the power of long distance educational programmes. Similar laboratories are currently being set up in Kenya and Zambia.

Phosphatidylinositol 3-kinase inhibition enhances human sperm motility and sperm-zona pellucida binding.

Various signalling pathways are involved in the regulation of sperm motility, capacitation, acrosome reaction and sperm-zona binding. Recent data pointed out an important role for phosphatidylinositol 3-kinase (PI3K) in human sperm motility. However, no study as of yet has been carried out to determine the effect of sperm treatment with the PI3K inhibitor LY294002 on other sperm parameters. In the present study, we investigated the role of PI3K on human sperm motility, acrosome reaction and sperm-oocyte binding by using this inhibitor. We demonstrate that in vitro incubation of washed unselected spermatozoa with LY294002 increased the percentage motility and progressive motility in asthenozoospermia patients as evaluated by computer-aided sperm analysis. The compound furthermore did not influence the acrosome reaction, whilst it (further) slightly enhanced sperm-oocyte binding. Our results therefore imply that PI3K negatively affects sperm motility and oocyte binding and might suggest a possible therapeutic role for PI3K inhibitors in the treatment regime for asthenozoospermia.

Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding.

Mammalian sperm must undergo a process known as capacitation before fertilization can take place. A key intracellular event that occurs during capacitation is protein tyrosine phosphorylation. The objective of this study was to investigate and visualize protein tyrosine phosphorylation patterns in human sperm during capacitation and interaction with the zona pellucida. The presence of specific patterns was also assessed in relation to the fertilizing capacity of the spermatozoa after in vitro fertilization. Protein tyrosine phosphorylation was investigated by immunofluorescence. Phosphorylation increased significantly with capacitation and was localized mainly to the principal piece of human sperm. Following binding to the zona pellucida, the percentage of sperm with phosphotyrosine residues localized to both the neck and the principal piece was significantly higher in bound sperm than in capacitated sperm in suspension. When the percentage of principal piece-positive sperm present after capacitation was <7%, fertilization rates after in vitro fertilization were reduced. Different compartments of human spermatozoa undergo a specific sequence of phosphorylation during both capacitation and upon binding to the zona pellucida. Tyrosine phosphorylation in the principal and neck piece may be considered a prerequisite for fertilization in humans.

Zona pellucida induced acrosome reaction, sperm morphology, and sperm-zona binding assessments among subfertile men.

PURPOSE: The study aimed to evaluate the relationship between the zona pellucida induced acrosome reaction (ZIAR) and (i) percentage normal spermatozoa as well as (ii) sperm-zona pellucida binding potential among men referred for a routine semen analysis. METHODS: Semen samples of 164 consecutive men referred to the andrology laboratory for routine semen analysis were studied. Semen samples were analyzed using the new WHO standards (strict criteria). ZIAR was recorded with a lectin conjugated Pisum sativum agglutinin microassay, while sperm-zona binding was evaluated with a standard hemizona assay (HZA). RESULTS: Andrology patients were divided according to the percentage normal spermatozoa in the ejaculate, namely <4% normal forms (n = 71), 5-14%, normal forms (n = 73), and >14% normal forms (n = 20). ZIAR data of the <4%, 5-14%, and >14% groups was (9.6 +/- 0.6)%, (13.9 +/- 0.5)%, and (15.0 +/- 1.1)%, respectively. The ZIAR data of fertile control men was (26.6 +/- 1.4)% which differed significantly from the three andrology referrals groups. Likewise significant differences were recorded during the hemizona assay namely, 38.0% (<4% normal forms), 54.5% (5-1% normal forms), and 62.6% (>14% normal forms). Among the group with >14% normal forms, five cases had impaired ZIAR outcome (<15%). Three of these men had normal morphology and HZAs. CONCLUSIONS: ZIAR testing should become part of the second level of male fertility investigations, i.e., sperm functional testing, since 15% of andrology referrals revealed an impaired acrosome reaction response to solubilized zona pellucida.