Globozoospermia.

Male infertility may be due to low sperm concentration, poor sperm motility, or abnormal morphology. Among the factors involved in male infertility, there is a rare morphology disorder called "Globozoospermia". This condition is primarily characterized by the presence of round-headed spermatozoa, absence of acrosomal cap and cytoskeleton defects around the nucleus. The morphological characteristics of globozoospermia are formed during spermiogenesis. We report here a case of male infertility due to morphological disorder Globozoospermia. Assessment of semen by observing macroscopic and microscopic parameters are not sufficient for sperm analysis. In present case, macroscopic and microscopic assessment was within normal range. Morphological assessment showed 80% of spermatozoa with round head and absence of acrosomal cap. The absence of acrosome makes fertilization impossible since these sperm are unable to bind to the zona pellucida. By using Intracytoplasmic Sperm Injection (ICSI), conception is possible; however, the fertilization rate remains very low.

Effect of bacterial infection on sperm quality and DNA fragmentation in subfertile men with Leukocytospermia.

BACKGROUND: Although bacterial infections have been recognized as a possible cause of male infertility, the effect of bacterial infections on sperm quality and sperm DNA fragmentation remains controversial. The current study aimed to investigate the prevalence rate of bacterial infection in subfertile men and its effect on semen quality. Seminal fluid was collected from 172 male members of infertile couples attending the andrology infertility center and a group of 35 fertile subjects as a control. Sperm parameters and DNA fragmentation were evaluated based on the type of bacteria in all ejaculates. RESULTS: From the 172 patients investigated for infertility, 60 (34.88%) patients had a positive culture for pathogenic bacteria of different species. Leukocytospermia was significantly higher in infected samples in comparison with non-infected samples (p < 0.05). Sperm concentration and motility and morphology were significantly lower in infected than non-infected samples. Moreover, sperm DNA fragmentation was significantly higher in infected than non-infected samples. Besides, our results showed that sperm DNA fragmentation was correlated significantly with leukocytospermia (R: 0.22, p < 0.01). CONCLUSION: The present study suggested that bacterial infection significantly correlated with leukocytospermia could impair male fertility potential through decreasing sperm motility, morphology, and DNA integrity.

One potential biomarker for teratozoospermia identified by in-depth integrative analysis of multiple microarray data.

Teratozoospermia is a common category of male infertility and with the increase in clinical patients and the increasing sophistication of assisted reproductive technology, there is an urgent need for an accurate semen diagnostic biomarker to accomplish rapid diagnosis of patients with teratozoospermia and accurately assess the success rate of assisted reproductive technologies. In this study, we performed gene differential expression analysis on two publicly available DNA microarray datasets (GSE6872 and GSE6967), followed by GSEA analysis to parse their enriched KEGG pathways, and WGCNA analysis to obtain the most highly correlated modules. Subsequent in-depth comparative analysis of the modules screened into the two datasets resulted in a gene set containing the identical expression trend, and then the differentially expressed genes in the set were screened using the corresponding criteria. Finally, three differentially expressed genes common to both datasets were selected. In addition, we validated the expression changes of this gene using another dataset (GSE6968) and in vitro experiments, and only screened one potential semen biomarker gene whose expression trend was identical to those in other datasets, which will also provide an important theoretical basis for the diagnosis and treatment of teratozoospermia.

Effect of Sperm Morphology in Intrauterine Insemination: Analysis of 115 Cycles and Literature Review.

IMPORTANCE: The value of morphology as a sperm parameter remains uncertain. Many studies have addressed the importance of morphology to predict the success of intrauterine insemination (IUI), but with conflicting results. OBJECTIVE: The aims of this study were to review the current literature, to query our own clinical experience via a retrospective, descriptive study, and to determine whether the diagnosis of isolated teratozoospermia influences pregnancy rate after IUI. RESULTS: We identified a large number of studies addressing this question. All were retrospective and most used different criteria to assess sperm morphology. Further complicating matters, the cutoff for normal morphology decreased from 15% to 4%. In our patient population, we found 12 cases of isolated teratozoospermia (10.43%). Only one of these produced an ongoing pregnancy and live birth. In all other cases, alteration of other sperm parameters coexisted (89.57%). These cycles produced a pregnancy rate of 13%, a nonsignficant difference. Pregnancy rates also were analyzed according to the percentage of normal morphology: 35.71% for less than 4%, 50% for 5% to 9%, and 14.29% for 10% to 14%. These rates did not differ significantly. CONCLUSIONS AND RELEVANCE: No consistent effect of sperm morphology on pregnancy rate was found in either the published literature or our own clinical experience. Larger and prospective studies are needed to identify any subtle effects of morphology on IUI outcomes that might exist.

MicroRNAs association with azoospermia, oligospermia, asthenozoospermia, and teratozoospermia: a systematic review.

Infertility is a major health problem across the world. One of the main reasons for male infertility are defects in sperm. Semen analysis is the most common test utilized to evaluate male fertility and since it suffers from multiple drawbacks, reproduction scientists have tried to find new molecular markers for detecting sperm defects. MicroRNAs (miRNAs) are small molecules in cells which take part in regulating gene expression. Various studies have confirmed miRNAs to have a role in defining multiple sperm characteristics, including sperm count, motility, and morphology. In this paper, we have systematically reviewed the role of miRNAs in infertile men with sperm defects including azoospermia, oligospermia, asthenozoospermia, and teratozoospermia. Also, we have assembled various bioinformatics tools to come up with a pipeline for predicting novel miRNAs which could possibly participate in sperm count, motility, and morphology. Also, related KEGG and GO terms for predicted miRNAs have been included in order to highlight their role in sperm function. Our study emphasizes the potential role of miRNAs in male infertility and provides a general overview for future studies aiming to find robust molecular markers for this condition.

Metabolic profiling of seminal plasma from teratozoospermia patients.

Teratozoospermia is one of conditions that can cause male infertility. The mechanism of teratozoospermia remains unclear. The knowledge of the metabolites in human seminal plasma (HSP) is meaningful for the pathological study of teratozoospermia. Analysis of changed metabolites in HSP can help understand the cellular mechanism, find the novel biomarkers and subsequently design a diagnosis test. In this study, the analysis of samples performed by proton nuclear magnetic resonance spectroscopy (1H NMR spectroscopy) to identify the various metabolites, with the aim of finding metabolic profiles and biomarkers related to male infertility. Eighteen de-regulated metabolites were identified in fertile men compared to teratozoospermia patients. These changes illustrate the deficiencies in absorption or metabolism of these metabolites in teratozoospermia. Furthermore, metabolic profiling showed that it is not possible to classify teratozoospermia based on teratozoospermia index (TZI). To the best of our knowledge, this is the first metabolic profiling analysis of HSP described the metabolic features of teratozoospermia in a holistic view.

The use of fluorescence in situ hybridization analysis on sperm: indications to perform and assisted reproduction technology outcomes.

PURPOSE: To determine the consequences of an altered sperm fluorescence in situ hybridization (FISH) result for ART outcomes and the indications for a sperm FISH analysis. METHODS: Data from 439 infertile men were collected. Bivariate analyses were performed to determine the association of men's age, seminal alterations, and sperm FISH indication, with the incidence of X, Y, 13, 18, and 21 sperm chromosomal abnormalities. A multivariate logistic regression analysis was performed to establish the most predictive variables for altered sperm FISH. Results from the IVF/ICSI cycles were collected for 248 out of 439 patients. Two distinct groups were established: 151 couples that used their own oocytes and 97 couples involved in egg donation programs. In both groups, ART outcomes were compared between normal and altered sperm FISH. RESULTS: Teratozoospermia and oligozoospermia were associated with sperm chromosome anomalies (p < 0.05). Indications for sperm FISH analysis with the highest predictability were teratozoospermia, male age, oligozoospermia, and implantation failure (AUC = 0.702). Embryo quality (p = 0.096), pregnancy rate (p = 0.054), and implantation rate (p = 0.089) were higher in own-oocytes couples with normal sperm FISH than in altered sperm FISH couples, although differences were not statistically significant. In donor-oocytes couples, in which high-quality embryos were transferred later than in own-oocytes couples (3.8 vs. 3.0 days), we did not identify differences in the ART outcome between normal and altered sperm FISH couples. In both groups, the possible interference of woman age was negligible. CONCLUSIONS: Sperm FISH is indicated in middle-aged oligoteratozoospermic patients with implantation failures in previous IVF/ICSI cycles. Sperm chromosome anomalies have a moderate detrimental impact on embryo quality, implantation, and pregnancy rates.

A novel homozygous FBXO43 mutation associated with male infertility and teratozoospermia in a consanguineous Chinese family.

OBJECTIVE: To identify the genetic causes of male infertility characterized by teratozoospermia. DESIGN: Genetic studies. SETTING: Medical university. PATIENT(S): Two infertile brothers with teratozoospermia in a consanguineous Chinese family, another 124 sporadic infertile male patients presenting with teratozoospermia, and 200 male controls with normal fertility. INVENTION(S): None. MAIN OUTCOME MEASURE(S): Whole exome sequencing and genotype analysis to identify the potential pathogenic mutation, Sanger sequencing to validate the mutation in family members, in silico structural modeling to predict the functional consequences of mutation, and targeted next-generation sequencing to validate the mutation in sporadic cases. RESULT(S): A novel homozygous nonsynonymous mutation (C1991T, p.G664D) in FBXO43 (F-box only protein 43) was observed in two brothers from a consanguineous Chinese family. The mutation was segregated with the disease phenotype and was predicted to be a disease causing protein by SIFT, PolyPhen-2, and Mutation Taster. An in silico mutant FBXO43 model predicts that the mutation p.G664D causes shortening of two ß-sheets, an additional α-helix, and change in loops, which may result in loss of function of the protein. The homozygous mutation of FBXO43 was absent in the 1000 Genomes Project (1000 G) and the Exome Aggregation Consortium (ExAC) databases. Subsequent mutation screening of FBXO43 in a cohort of 124 cases identified four additional cases with heterozygous FBXO43 mutations. No mutations were found in FBXO43 in 200 fertile controls. CONCLUSION(S): The mutation in FBXO43 is a causative factor of male infertility and teratozoospermia.

Biallelic mutations in PMFBP1 cause acephalic spermatozoa.

The majority of men with defects in spermatogenesis remain undiagnosed. Acephalic spermatozoa is one of the diseases causing primary infertility. However, the causes underlying over half of affected cases remain unclear. Here, we report by whole-exome sequencing the identification of homozygous and compound heterozygous truncating mutations in PMFBP1 of two unrelated individuals with acephalic spermatozoa. PMFBP1 was highly and specifically expressed in human and mouse testis. Furthermore, immunofluorescence staining in sperm from a normal control showed that PMFBP1 localizes to the head-flagella junction region, and the absence of PMFBP1 was confirmed in patients harboring PMFBP1 mutations. In addition, we generated Pmfbp1 knock-out (KO) mice, which we found recapitulate the acephalic sperm phenotype. Label-free quantitative proteomic analysis of testicular sperm from Pmfbp1 KO and control mice showed 124 and 35 proteins, respectively, increased or decreased in sperm from KO mice compared to that found in control mice. Gene ontology analysis indicates that the biological process of Golgi vesicle transport was the most highly enriched in differentially expressed proteins, indicating process defects related to Golgi complex function may disturb formation of the head-neck junction. Collectively, our data indicate that PMFBP1 is necessary for sperm morphology in both humans and mice, and that biallelic truncating mutations in PMFBP1 cause acephalic spermatozoa.

Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella.

STUDY QUESTION: Can whole-exome sequencing (WES) of infertile patients identify new genes responsible for multiple morphological abnormalities of the sperm flagella (MMAF)? SUMMARY ANSWER: WES analysis of 78 infertile men with a MMAF phenotype permitted the identification of four homozygous mutations in the fibrous sheath (FS) interacting protein 2 (FSIP2) gene in four unrelated individuals. WHAT IS KNOWN ALREADY: The use of high-throughput sequencing techniques revealed that mutations in the dynein axonemal heavy chain 1 (DNAH1) gene, and in the cilia and flagella associated protein 43 (CFAP43) and 44 (CFAP44) genes account for approximately one-third of MMAF cases thus indicating that other relevant genes await identification. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of 78 patients presenting a MMAF phenotype who were recruited in three fertility clinics between 2008 and 2015. Control sperm samples were obtained from normospermic donors. Allelic frequency for control subjects was derived from large public databases. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES was performed for all 78 subjects. All identified variants were confirmed by Sanger sequencing. Relative mRNA expression levels for the selected candidate gene (FSIP2) was assessed by quantitative RT-PCR in a panel of normal human and mouse tissues. To characterize the structural and ultrastructural anomalies present in patients' sperm, immunofluorescence (IF) was performed on sperm samples from two subjects with a mutation and one control and transmission electron microscopy (TEM) analyses was performed on sperm samples from one subject with a mutation and one control. MAIN RESULTS AND THE ROLE OF CHANCE: We identified four unrelated patients (4/78, 5.1%) with homozygous loss of function mutations in the FSIP2 gene, which encodes a protein of the sperm FS and is specifically expressed in human and mouse testis. None of these mutations were reported in control sequence databases. TEM analyses showed a complete disorganization of the FS associated with axonemal defects. IF analyses confirmed that the central-pair microtubules and the inner and outer dynein arms of the axoneme were abnormal in all four patients carrying FSIP2 mutations. Importantly, and in contrast to what was observed in patients with MMAF and mutations in other MMAF-related genes (DNAH1, CFAP43 and CFAP44), mutations in FSIP2 led to the absence of A-kinase anchoring protein 4 (AKAP4). LIMITATIONS, REASONS FOR CAUTION: The low number of biological samples and the absence of a reliable anti-FSIP2 antibody prevented the formal demonstration that the FSIP2 protein was absent in sperm from subjects with a FSIP2 mutation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that FSIP2 is one of the main genes involved in MMAF syndrome. In humans, genes previously associated with a MMAF phenotype encoded axonemal-associated proteins (DNAH1, CFAP43 and CFAP44). We show here that FSIP2, a protein of the sperm FS, is also logically associated with MMAF syndrome as we showed that it is necessary for FS assembly and for the overall axonemal and flagellar biogenesis. As was suggested before in mouse and man, our results also suggest that defects in AKAP4, one of the main proteins interacting with FSIP2, would induce a MMAF phenotype. Finally, this work reinforces the demonstration that WES sequencing is a good strategy to reach a genetic diagnosis for patients with severe male infertility phenotypes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 (14-CE15) and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.

System analysis of teratozoospermia mRNA profile based on integrated bioinformatics tools.

mRNA has an important role in spermatogenesis and the maintenance of fertility, and may act as a potential biomarker for the clinical diagnosis of infertility. In the present study, potential biomarkers associated with teratozoospermia were screened through systemic bioinformatics analysis. Initially, genome­wide expression profiles were downloaded from the Gene Expression Omnibus and primary analysis was conducted using R software, which included preprocessing of raw microarray data, transformation between probe ID and gene symbol and identification of differentially expressed genes. Subsequently, a functional enrichment analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery to investigate the biological processes involved in the development of teratozoospermia. Finally, a protein­protein interaction network of notable differentially expressed genes was constructed and cross­analysis performed for multiple datasets, to obtain a potential biomarker for teratozoospermia. It was observed that G protein subunit ß 3, G protein subunit α o1 and G protein subunit g transducin 1 were upregulated and enriched using Kyoto Encyclopedia of Genes and Genomes (KEGG) in the network and in cross analysis. Furthermore, ribosomal protein S3 (RPS3), RPS5, RPS6, RPS16 and RPS23 were downregulated and enriched using KEGG in teratozoospermia. In conclusion, the results of the present study identified several mRNAs involved in sperm morphological development, which may aid in the understanding and treatment of infertility.

Preparation and observation methods can produce misleading artefacts in human sperm ultrastructural morphology.

The aim of this study was to evaluate the production of artefacts during preparation and observation of human sperm for ultrastructural morphology and analyse the possible reasons of causing these artefacts. Under the scanning electron microscopy, damaged sperm heads (crack or/and rupture), necks (head-neck or head-midpiece separation) and midpiece (disassembled and denuded axoneme ultrastructures, bent midpiece) were analysed to be the consequence of exogenous effects. Thirty infertile men with teratozoospermia revealed more spermatozoa with damage to head, neck and midpiece than did thirty fertile males (p < .01). After the samples from fertile males underwent five repeated observations, most sperm heads and necks in the samples were destroyed when compared with the single observation (p < .01). Destroyed sperm heads were full of cracks and peelings, even sperm tails were broken and fragmented, and separations of the sperm head-neck or head-midpiece became common. Spermatozoa from fertile males with centrifugation of 600 g for washing sperm exhibited more damage to the midpiece than those with the 300 g (p < .01). These results demonstrate that preparation and observation methods can damage sperm ultrastructures, leading to producing artefacts of ultrastructural morphology. The artefacts of sperm ultrastructural morphology may be associated with sperm structural fragility, preparation conditions and electron beam damage.

Testing for genetic contributions to infertility: potential clinical impact.

INTRODUCTION: Male infertility affects about 7% of the general male population, and it is a multifactorial, polygenic pathological condition. Known genetic factors, accounting for about 20-25% of male factor infertility, are present in each etiological category: i) hypothalamic-pituitary axis dysfunction; ii) quantitative and qualitative alterations of spermatogenesis; iii) ductal obstruction/dysfunction. Areas covered: All routinely available genetic tests are described. Indication for testing for chromosomal anomalies and Y chromosome microdeletions is based on sperm count (severe oligozoospermia/azoospermia). Mutation screening in candidate genes is indicated in specific semen/testis phenotypes. In about 40% of infertile patients, the aetiology remains unknown ('idiopathic cases') and whole exome sequencing may reveal novel genetic causes. Expert commentary: Genetic testing is essential for its relevance in clinical decision-making. For instance, it helps to avoid unnecessary surgical or medical treatments and it may provide prediction for testicular sperm retrieval. The highest frequency of genetic anomalies is observed in severe spermatogenic impairment, which can be treated with in vitro fertilization (IVF). Given the risk of transmitting genetic disorders to the future offspring through IVF, the diagnosis of known and the discovery of novel genetic factors in idiopathic infertility is of outmost clinical importance.

Male infertility problems of patients with strict sperm morphology between 5-14% may be missed with the current WHO guidelines.

OBJECTIVE: In 2010 W.H.O. changed the lower reference limit for strict sperm morphology from 15 to 4%. The change was based on 5th percentile cut points from a meta-analysis on a published series of fertile men. This study investigates if patients referred for evaluation with sperm morphologies between 5-14% have identifiable etiologies of male infertility. MATERIALS AND METHODS: I.R.B. approval was obtained to review records for patients referred to the University of Michigan Center of Reproductive Medicine between May 2012-May 2014 whom had a sperm morphology of 5-14%. Semen analysis, hormone levels, and information related to an infertility diagnosis, were recorded into a de-identified database. Patients were placed into the categories 'Varicocele', 'Hypogonadism', 'Intercourse problems', 'Anti-sperm antibodies (A.S.A.)', 'Other' or 'No diagnosis'. RESULTS: A total of 253 patients were included in the study. Of these, 96/253 (38%) had a clinical varicocele; 44/253 (17%) hypogonadism; 4/253 (2%) intercourse problems; 11/253 (4%) evidence of sperm antibodies; and 15/253 (6%) had various other problems deemed potentially contributing causes of infertility. In all, nearly 67% of the subjects were identified to have a potential contributing etiology of male infertility. Similar results were found for the men with isolated low morphology (n = 194). CONCLUSIONS: This study demonstrates that 67% of men in infertile couples, who have strict sperm morphology between 5 and 14%, are found to have a potential contributing male factor infertility diagnosis. This raises the possibility that the new lower reference value for sperm morphology may result in missed opportunities for proper infertility assessment.

The effect of bacteriospermia and leukocytospermia on conventional and nonconventional semen parameters in healthy young normozoospermic males.

Bacterial semen inflammation/infection is an important diagnostic and therapeutic problem in contemporary andrology. The molecular mechanism by which inflammatory mediators compromise the fertilizing potential of germ cells is complex and multifactorial, and it remains unclear. To improve the understanding of the pathophysiology of human subfertility/infertility caused or complicated by reproductive tract inflammation/infection, we simultaneously evaluated a set of conventional (standard semen analysis) and nonconventional sperm parameters, including subcellular changes in sperm membranes (phospholipid scrambling, peroxidative damage, and phosphatidylserine (PS) externalization), mitochondria (mitochondrial transmembrane potential, ΔYm, and oxidoreductive capability), and DNA fragmentation in healthy young normozoospermic males with asymptomatic bacteriospermia and leukocytospermia. Both bacteriospermia and leukocytospermia had a deleterious effect on standard sperm parameters, including sperm concentration, motility and morphology. Bacteriospermia was associated with a simultaneous decrease in mitochondrial transmembrane potential and an increase in PS externalization, and with DNA fragmentation in both live and dead sperm. The highest MDA concentrations in sperm lysates were observed in the presence of leukocytes. This study demonstrates for the first time that bacteriospermia and leukocytospermia compromise sperm quality in healthy young normozoospermic males. Bacteria mainly participate in intrinsic mitochondria-dependent apoptotic cell death mechanisms. Oxidative stress plays a relevant role in decreasing routine sperm parameters during leukocytospermia. The value of these observations may be significant and may support the development of a new diagnostic platform (biomarkers) for infertile males with infections in the reproductive tract.

Systematic tracking of altered modules identifies disrupted pathways in teratozoospermia.

The objective of this study was to identify disrupted pathways in teratozoospermia by systematically tracking dysregulated modules in reweighted protein-protein interaction (PPI) networks. We inferred and reweighted the PPI networks of normal and teratozoospermia groups based on Spearman correlation coefficients. Modules in the PPI networks were explored via a clique-merging algorithm and altered modules were identified based on maximum weight bipartite matching. Furthermore, pathway-enrichment analyses of genes in altered modules were performed by Database for Annotation, Visualization, and Integrated Discovery (DAVID) to illuminate the biological pathways in teratozoospermia. A total of 20,102 genes were screened from the expression profile. We explored 2406 and 2101 modules in normal and disease PPI networks, respectively. Moreover, we obtained 875 altered modules by comparing modules in normal and teratozoospermia PPI networks. At P < 0.01, the genes involved in 2855 interactions with score changes >1 were mainly enriched in 66 pathways and the genes in altered modules were enriched in 71 pathways. The activity genes (missed and added genes in the disrupted modules) were enriched in 41 common pathways. There were 36 mutual enriched pathways under the five different conditions. Moreover, the cell cycle pathway was disrupted in the first 10 pathways of each condition. This study provides a powerful biomarker discovery platform to better understand the progression of teratozoospermia by systematically tracking dysregulated modules. This method uncovered potential diagnostic and therapeutic targets of teratozoospermia. This information might lead to improved monitoring and treatment of teratozoospermia.

Decreased melatonin levels and increased levels of advanced oxidation protein products in the seminal plasma are related to male infertility.

Melatonin, an indolamine secreted by the pineal gland, is known as a powerful free-radical scavenger and wide-spectrum antioxidant. Therefore, the aim of this study was to correlate markers of oxidative protein damage (advanced oxidation protein products, AOPPs) and the total antioxidant capacity (TAC) with melatonin levels in the seminal plasma of men with azoospermia (n=37), theratozoospermia (n=29) and fertile controls (normozoospermia, n=37). Melatonin concentration was measured by radioimmunoassay. The levels of AOPP as well as TAC efficiency (determined by the ferric reducing antioxidant power, FRAP) were estimated by spectrophotometric methods. The concentration of melatonin and AOPP significantly differed in azoospermic (P<0.0001) and theratozoospermic (P<0.0001) patients versus fertile men, and correlated negatively (r=-0.33, P=0.0016). The TAC levels were significantly higher in azoospermia than in theratozoospermia (P=0.0022) and the control group (P=0.00016). In azoospermia, the AOPP concentration was also significantly higher than that observed in theratozoospermia (P=0.00029). Decreased levels of melatonin together with elevated AOPP altered the oxidative-antioxidative balance in the ejaculate, thereby reducing fertility. Therefore, melatonin and AOPP levels may serve as additional diagnostic markers of semen quality and male reproductive potential.

A new technique for analysis of human sperm morphology in unstained cells from raw semen.

Sperm morphology analysis is a fundamental component of semen analysis, but its real significance has been clouded by the plethora of techniques used for its evaluation. Most involve different fixation and staining procedures that induce artefacts. Herein we describe Trumorph (Proiser R+D, Paterna, Spain), a new method for sperm morphology analysis based on examination of wet preparations of spermatozoa immobilised, after a short 60°C shock, in narrow chambers and examined by negative phase contrast microscopy. A range of morphological forms was observed, similar to those found using conventional fixed and stained preparations, but other forms were also found, distinguishable only by the optics used. The ease of preparation makes the Trumorph a robust method applicable for the analysis of living unmodified spermatozoa in a range of situations. Subsequent studies on well-characterised samples are required to describe the morphology of spermatozoa with fertilising potential.