[Show abstract][Hide abstract] ABSTRACT: Whole arm t(9;13)(p11;p12) translocations are rare and have been described only a few times; all of the previously reported cases were familial.
We present here an infertile male carrier with a whole-arm reciprocal translocation dic(9;13)(p11.2;p12) revealed by GTG-, C-, and NOR-banding karyotypes with no mature sperm cells in his ejaculate. FISH and genome-wide 400 K CGH microarray (Agilent) analyses demonstrated a balanced chromosome complement and further characterised the abnormality as a dicentric chromosome (9;13): dic(9;13)(pter[rightwards arrow]p11.2::p12[rightwards arrow]qter),neo(9)(pter[rightwards arrow]p12[rightwards arrow]neo[rightwards arrow]p11.2). An analysis of the patient's ejaculated cells identified immature germ cells at different phases of spermatogenesis but no mature spermatozoa. Most (82.5%) of the germ cells were recognised as spermatocytes at stage I, and the cell nuclei were most frequently found in pachytene I (41.8%). We have also undertaken FISH analysis and documented an increased rate of aneuploidy of chromosomes 15, 18, X and Y in the peripheral blood leukocytes of our patient. To study the aneuploidy risk in leukocytes, we have additionally included 9 patients with non-obstructive azoospermia with normal karyotypes.
We propose that the azoospermia observed in the patient with the dic(9;13)(p11.2;p12) translocation was most likely a consequence of a very high proportion (90%) of association between XY bivalents and quadrivalent formations in prophase I.
[Show abstract][Hide abstract] ABSTRACT: To identify potential biomarkers of azoospermia to determine a particular stage of spermatogenetic differentiation.
GeneChip Human Gene 1.0 ST microarray with validation at mRNA and protein levels.
Basic research laboratory.
Men with various types of nonobstructive azoospermia (n = 18) and with normal spermatogenesis (n = 4).
Obtaining 31 testicular biopsy samples.
Gene expression analysis using the Affymetrix Human Gene 1.0 ST microarrays on 14 selected genes according to the highest fold change, verified with quantitative polymerase chain reaction and on independent set of microarray samples. Western blot and immunohistochemistry were additionally performed.
The comparative analysis of gene expression profiles in the infertile and control groups resulted in the selection of 4,946 differentially expressed genes. AKAP4, UBQLN3, CAPN11, GGN, SPACA4, SPATA3, and FAM71F1 were the most significantly down-regulated genes in infertile patients. Global analysis also led to identification of up-regulated genes-WBSCR28, ADCY10, TMEM225, SPATS1, FSCN3, GTSF1L, and GSG1-in men with late maturation arrest. Moreover, the results from quantitative polymerase chain reaction and Western blot largely confirmed the microarray data.
The set of selected genes can be used to create a molecular diagnostic tool to determine the degree of spermatogenic impairment for men with idiopathic nonobstructive azoospermia.
Fertility and sterility 09/2013; · 4.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to examine the expression levels of IL-1 family members, IL-6, IL-10, TNF family, SCF, and c-kit in infertile patients with idiopathic non-obstructive azoospermia (NOA) compared with men with normal spermatogenesis.
We analyzed 20 testicular biopsy samples with Affymetrix Human Gene 1.0 ST microarrays (Affymetrix, Santa Clara, CA, USA). Sixteen of them were obtained from patients with various types of NOA and four with normal spermatogenesis.
The comparative analysis of normal and pathological group demonstrated a different expression level of IL1-RA gene. It was also observed that the gene expression levels for IL1-R1, CASP1, and stem cell factor (SCF) were upregulated in the Sertoli-cell-only syndrome group in comparison with the control one (P < 0.05).
The microarray analysis showed the expression level of all investigated paracrine/autocrine factors at one go, and therefore, the possible interaction between these genes could be examined.
American Journal Of Reproductive Immunology 07/2013; · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: The Nanos gene is a key translational regulator of specific mRNAs involved in Drosophila germ cell development. Disruption of mammalian homologues, Nanos2 or Nanos3, causes male infertility in mice. In humans, however, no evidence of NANOS2 or NANOS3 mutations causing male infertility has been reported. Although Nanos1 seems dispensable for mouse reproduction, we sought to analyse for the first time its homologue in infertile men. METHODS: A group of 195 patients manifesting non-obstructive azoospermia or oligozoospermia were tested for mutations of the NANOS1 gene, using single-strand conformation polymorphism and DNA sequencing. RESULTS: Three types of NANOS1 gene mutations were identified in five patients and were absent in 800 chromosomes of fertile men. Pedigree analysis indicated a dominant inheritance pattern with penetration limited to males. Two mutations caused deletions of single amino acids, p.Pro77_Ser78delinsPro and p.Ala173del, each of them identified in two unrelated patients. Both types of deletions were located in the NANOS1 N-terminus (responsible for protein interactions) and were associated with a lack of germ cells in testes. Interestingly, the Pro77_Ser78delinsPro mutation altered interaction of NANOS1 with a microRNA biogenesis factor, GEMIN3. The third identified mutation, p.[(Arg246His; Arg276Tyr)], found in the C-terminal RNA-binding domain, was present in a single oligo-astheno-teratozoospermic man. We bioinformatically demonstrated that the p.Arg246His substitution causes a decrease in the positive charge of this domain, potentially altering RNA-binding. CONCLUSIONS: This is the first report describing the association of NANOS1 gene mutations with human infertility. Two different infertility phenotypes may reflect distinct functions of N-terminal versus C-terminal regions of NANOS1.
Journal of Medical Genetics 01/2013; · 5.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Semen analysis is an important part of male infertility diagnosis and should be performed according to international recommendations. The reliability of the results depends mainly on the qualifications of the laboratory analysts. External Quality Assessment Programmes (EQAP) are performed in laboratories worldwide in order to standardize the results. The aim of this study was to perform the first in Poland comparison of the results of sperm analysis from different laboratories.
Forty two Polish laboratories were invited to participate in the EQAP and eight laboratories agreed to take part in the analysis. They were sent uniform semen samples, prepared in accordance with the WHO standards: one sample for the assessment of concentration, two for motility (on DVD) and two for the morphology of the sperm (Papanicolau staining). The reference group was comprised of three employees of the Andrologic Laboratory of Poznań University of Medical Sciences, who regularly take part in EQAP organized by ESHRE. The reference group analyzed the samples from the participating laboratories. Statistic features such as mean, median, standard deviation, minimal and maximal value, first and third quartile were assessed for every examined parameter Z-score index was used to compare the differences between assessed laboratories and the reference laboratory. The acceptable Z-score range was +/-1.
Average concentration in the reference group was 43 mln/ml, while in the assessed laboratories (L) it was between 31 (L4) and 72.5 mln/ml (L1). Z-score for concentration analysis in three laboratories exceeded +/- 1 (2.51 for L1, 1.42 for L2 and -1.02 for L4). In the analysis of the first sample for progressive motility the reference group received 59%, while the values of participating laboratories varied from 42,5% (L1) to 80% (L4). Most of the centers achieved Z-score within the normal range, except for L1 and L4 (-1.48 and 1.88 respectively). The reference value for the second sample for motility was 33%. The received results ranged between 22% (L6) and 48% (L7). Z-score was -1.27 for L6 and 1.73 for L7, while the results of other laboratories were close to the reference group. Reference morphology for the first sample was 3%. The results sent by the participating laboratories varied from 2% (L2 and L3) to 72.7% (L8). Assessment of the first sample of sperm morphology resulted in significant differences in 4 laboratories: Z-score of 12.41 for L8, 2.54 for L5, 2.19 for L4 and 1.12 for L1. The reference morphology for the second sample was 1%, while the sent results ranged from 0% (L3) to 45.8% (L8). Z-score for the second sample was 5.69 for L8, and 3.07 for L4 and 1.18 for L5.
1) Significant differences between the laboratories in the obtained results of the analysis of sperm parameters, especially the morphology were found during the external quality assessment of the semen analysis. 2) Taking part in EQAP provides laboratories with information which procedures and stages ought to be improved in order to increase the reliability of semen results.
Ginekologia polska 11/2012; 83(11):835-40. · 0.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since testes and adrenal cortex derive from the same urogenital ridge, adrenal tissue with descending gonads may migrate in early embryonic period. Although most often ectopic tissue undergoes atrophy, in some cases, when adrenocorticotrophic (ACTH) overstimulation occurs, the adrenal remnants in the testes may become hypertrophic and form testicular adrenal rest tumors (TARTs). The growth of TARTs in the testes leads to obstruction of the seminiferous tubules which can mechanically impair the function of the gonads and cause irreversible azoospermia. We describe a patient suffering since neonatal period from congenital adrenal hyperplasia (CAH), disorder with defected pathway of cortisol production, which leads to increased ACTH production and to overstimulation of adrenal cortex. He had very poor disease control and therefore in late puberty he was diagnosed with TARTs. At the age of 19.5 he was diagnosed with azoospermia, most likely caused by TARTs. It is the first evidence of TARTs in Polish literature. Although not many cases have been published so far, the incidence of TARTs seems to be highly underdiagnosed, so it seems reasonable to consider the disease in differential diagnosis of male infertility.
Ginekologia polska 09/2012; 83(09):700-702. · 0.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Around one million couples in Poland suffer from infertility People in reproductive age are most active Internet users. The aim of the study was to assess Internet habits of infertile patients. We checked to what extent infertile patients seek information about infertility on-line and what is their approach to the information found.
85 female patients treated for infertility for at least one year were surveyed. The anonymous questionnaire was designed by the authors of the publication. It consisted of questions related to medical history of the patients and sources of information about infertility they used. It also checked Internet activity of the patients and contained Beck's Depression Inventory (BDI). Chi-square test and Spearman's correlation test were used to evaluate the results.
The majority of patients used Internet to find information about infertility (93%); 46% of the respondent declared Internet forums to be their main source of information about it. Patients used on-line sources of information more often than stricte medical sources. Internet influenced their relation with the physician. 64% of patients verified on-line information and treatment proposed by their doctor before using them. One third of the surveyed women claimed their knowledge about infertility comes more from the Internet than the specialist who treated them. There was a positive correlation between patients who checked diagnostic or therapeutic methods proposed by their physician with depression in BDI.
Considering the great impact of Internet forums and web pages on patient approach to diagnostics and treatment of infertility there seems to be a need to create a professional Polish website and forum to provide the patients with reliable information about the disease.
Ginekologia polska 04/2012; 83(4):250-4. · 0.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protamine 1 (PRM1) and 2 (PRM2) are major nuclear proteins in spermatozoa known to bind to chromatin during early spermatogenesis. The aim of this study was to evaluate the impact of protamine transcripts and proteins on human spermatozoa and their fertilization ability as well as the development of preimplantation embryos. Oocytes selected from 92 patients were fertilized in vitro (IVF) using their partners' sperm after evaluating its concentration, motility and morphology. Reverse transcription and real-time quantitative PCR (RQ-PCR) were applied to determine protamine mRNA level, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were used to quantify the protamine protein concentration. Next, protamine mRNA and protein levels were correlated with sperm concentration, motility and morphology as well as with the fertilization ability of mature spermatozoa and embryos of different quality obtained from the IVF program. A significant correlation has been found between protamine transcripts/proteins and: sperm concentration, progressive sperm motility, sperm fertilization ability as well as embryo quality. Protamine transcripts and proteins contribute to the quality of spermatozoa, successful fertilization and may have a significant influence on the development of preimplantation embryos.
[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to assess the outcome of pregnancies after in vitro fertilization protocol (IVF), that were complicated by the ovarian hyperstimulation syndrome (OHSS).
We examined 26 women undergoing IVF due to OHSS. Patients were divided into two groups: with early OHSS and late OHSS. All women were screened for age, Body Mass Index (BMI), number of embryos transferred, OHSS symptoms and duration of hospitalization. Pregnancy outcome was assessed by rate of miscarriages, premature deliveries and multiple gestations. We assessed also the delivery mode, birth weight of all newborns and obstetric complications.
Early OHSS occurred in 10 patients and late OHSS complicated the pregnancies of 16 women. 94.1% of the late OHSS cases occurred in cycles with pregnancy. The miscarriage rate was 26.9%. 38.5% of all births were premature and 26.9% of the newborns had low birth weight. Multiple pregnancies were confirmed in 46.2% of the patients, triplets mainly in women with late OHSS. No fetal anomalies were diagnosed. The most common antenatal complications were: diabetes mellitus, cholestasis and intrauterine infection. The rate of the cesarean sections was 73.8%.
OHSS is a risk factor for miscarriage, preterm delivery multiple gestation, obstetric complications and cesarean section. Late OHSS may be closely associated with the conception cycles.
Ginekologia polska 02/2012; 83(2):104-10. · 0.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylserine membrane translocation (PST) is considered to be a marker of apoptosis; however, numerous studies have reported on its role in processes not related to cell death. The purpose of the study was to investigate: (1) what is the impact of PST on the motility of spermatozoa, and (2) does the swim-up isolation involve the percentage of cells presenting PST? Semen of 28 normozoospermic men (WHO criteria) was analyzed. High motility spermatozoa were isolated by the swim-up technique. The percentage of spermatozoa with PST in neat semen and after swim-up isolation was assessed with Annexin-V labeled with fluorescein, using flow cytometry technique. The spermatozoas' motility was measured with a computer-assisted analysis system. The kinetic subpopulations of spermatozoa were identified with dedicated software and analyzed regarding PST. Vital spermatozoa with PST demonstrated progressive movement. The motion analysis system revealed a very strong positive correlation between the percentage of vital spermatozoa with PST and the percentage of spermatozoa belonging to the slow subpopulation (r = 0.83; p < 0.05), as well as a very strong negative correlation between the percentage of vital spermatozoa with PST and the percentage of spermatozoa belonging to the rapid subpopulation (r = -0.86; p < 0.05). After the swim-up isolation, the percentage of vital spermatozoa presenting PST significantly decreased (2.4 ± 2.1% vs. 5.2 ± 2.4%; p < 0.05). Spermatozoa with PST present progressive movement; however, their motility is decreased. Isolation of spermatozoa with the swim-up technique eliminates the cells with PST.
[Show abstract][Hide abstract] ABSTRACT: The complex structure of the human spermatozoa membrane comprises five topographic domains. Transmembrane asymmetry of the distribution of phospholipids including phosphatidylserine (PS) is considered a marker of cell activity. The objective of the study was to determine which cytomembrane domains of human spermatozoa are involved in PS membrane translocation and to identify the possible relationship of PS translocation with spermatozoa morphology and vitality. In normozoospermic semen of 35 donors, annexin-V labeling with fluorescein determined PS translocation. Propidium iodide staining distinguished between vital and dead spermatozoa. Three types of PS membrane translocation have been distinguished: (1) in the midpiece, (2) in the acrosomal part and (3) simultaneously in the midpiece and acrosomal part. In morphologically normal vital spermatozoa, PS translocation occurred in the midpiece but never in the equatorial region. In dead spermatozoa, simultaneous PS translocation in the midpiece and acrosomal part was most often observed. The difference between proportions of, respectively, vital and dead spermatozoa presenting PS translocation located in different domains was significant (P < 0.0001). In vital cells, there was no difference in PS translocation prevalence between morphologically normal and abnormal spermatozoa (P > 0.05). The strict relation of PS translocation to specific membrane domains indicates functional specificity. It seems doubtful to include this phenomenon in physiological mechanisms of elimination of abnormal spermatozoa.
Journal of Membrane Biology 03/2011; 240(3):165-70. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infertility seems to be a great worldwide problem. Many publications present the epidemiology of infertility, but the percentage distribution of factors responsible for infertility varies significantly. The objective was to define infertility profiles in Poland assessed according to the information provided by 4 large infertility centers: Bialystok, Poznan, Szczecin and Warsaw.
Multicenter study was performed. Couples with primary infertility, attending one of the four centers in Poland, were asked to fulfill a questionnaire. Overall 1517 questionaires were analysed: Bialystok - 468, Poznan - 325, Szczecin - 341, Warsaw - 383. Only semen analyses fulfilling WHO Manual 1999 criteria were used in the study and were afterwards compared with 2010 WHO standards.
Results from 4 Polish centers showed that the average age of infertile women was 31.41 years and of infertile men 33.2 years. The mean duration of infertility equaled 3.31 years. Among 1517 surveyed women, no pathological findings concerning reproductive abilities were observed in 1088 cases (71.72%). In the remaining patients the following were diagnosed: uterine factor in 26 (6.02%) women, ovulation disorders in 134 (31.33%), including 70 (16.27%) of PCOS patients, tubal factor in 165 (38.55%) and endometriosis in 145 (33.73%). The average of 18.9% of couples had a mixed cause of infertility, while idiopathic factor was assigned to 15.99%. In the study male factor accounted for 55.73% of cases. Change of reference values for semen analysis implemented in 2010 caused an increase in the number of normal results and asthenozoospermia.
According to the data provided by Polish infertility centers the rate of male factor as a reason of infertility has reached 55.73%. However, the implementation of new reference values for semen analysis in 2010 led to the decrease in male factor frequency and the increase in the rate of idiophatic infertility. Anovulatory cycles and endometriosis are the main reasons affecting the female reproductive potential.
[Show abstract][Hide abstract] ABSTRACT: Androgen insensitivity syndrome (AIS, Morris syndrome) is an X-linked recessive disorder of sexual development caused by the mutation of the androgen receptor coding gene (locus Xq11-q12). We present a case of a 17-year-old girl diagnosed because of primary amenorrhea. Her cytogenetic analysis revealed the kariotype of 46, XY. Clinical examinations, based on the Quigley's scale, showed features of complete AIS. Hormonal tests brought the following results: FSH - 2.81 mIU/ml, LH - 13.88 mIU/ml. Testosterone value met the norm for a male individual in reproductive age (7.97 ng/ml). Family anamnesis revealed no episodes of genetic diseases. Due to the risk of neoplasia, the diagnosis was an indication for surgical gonads removal, which was performed laparoscopically. Histopathologic examination showed tubular adenoma with immature seminiferous tubules without spermatogenesis, and serous cysts. Patient was discharged on the next day after the operation in good general condition, and estrogen therapy was prescribed. CONCLUSION: Laparoscopy is an effective method in AIS treatment.
Ginekologia polska 06/2010; 81(6):457-60. · 0.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transcription levels of stem cell factor (SCF) and c-kit were examined using real-time RT PCR in interstitial and intratubular cell fractions, as well as in tissue homogenates from normal, azoospermic and neoplasmic patients. Peripheral blood mononuclear cells (PBMC) were used as a systemic control. The observed level of c-kit expression in all investigated groups was generally higher than the expression of SCF. The highest (statistically significant) level of c-kit was noted in testicular tumours (the greater part of which were represented by seminomas) in contrast to SCF mRNA, which may indicate an association between c-kit overexpression and seminoma development. In Sertoli cell only syndrome, almost equal levels of SCF and c-kit transcripts were noted. These results may indicate Leydig cells as the alternative source of c-kit gene transcription. SCF transcript values were low and comparable among the analysed subgroups except that in maturation arrest at spermatocyte stage, the SCF gene expression was statistically higher than in testicular tumours. It appears from the study that c-kit has been a dynamic gene, changing its activity in a variety of testicular pathologies while being expressed in all testicular compartments but clearly overexpressed in testicular tumours of seminomatous origin.
[Show abstract][Hide abstract] ABSTRACT: To investigate the expression of genes coding for selected cytokines with antagonistic functions (IL-6, IL-10, TNF-alpha) as well as TNF-alpha receptors (TNFR1 and TNFR2) in correct spermatogenesis (normal proliferation), maturation arrest (proliferation inhibited) and testicular tumors (overgrowth).
Transcription levels of genes coding for IL-6, IL-10, TNF-alpha, TNFR1 and TNFR2 were quantitatively examined using a real-time RT-PCR.
Significantly higher amounts of IL-6 mRNA were observed in testicular tumor samples than in normal spermatogenesis or in some syndromes with maturation arrest (MA at spermatid level or SCOS), while IL-10 gene levels were fairly stable. In homogenates with maturation arrest, the expression of TNFR1 gene was markedly higher than in testicular tumors, while the opposite phenomenon was found in respect to TNFR2 gene.
The results obtained indicate that changes in activities of intra-testicular cytokines may promote different distinct pathologies such as testicular cancer or infertility.
American Journal Of Reproductive Immunology 08/2009; 62(1):51-9. · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study was carried out to investigate the relationship between sperm subpopulation kinetics on in vitro fertilization rate. The ability of human sperm to achieve fertilization oocytes was investigated in relation to particular motility parameters obtained on a computer aided sperm analysis system base. Analysis covers velocity straight linear (VSL), cross beat frequency (CBF), lateral head displacement (LHD) and homogeneity of progressive motility velocity (HPMV) of fresh semen and semen after density gradient selection. Investigation was based on sperm samples from 82 infertile couples undergoing IVF. Two subpopulations were extracted from each sample using the clustering method with respect to VSL parameter: a slow and rapid one. Comparison of obtained results before and after selection shows no significant change of subpopulations percentage. However, this method of selection strongly influences motility parameters of both subpopulations. There was found a positive correlation for VSL, LHD and HPMV and a negative correlation for CBF parameters found in slow fraction of fresh semen and percentage of fertilized oocytes. On the other hand, rapid subpopulation parameters for fresh semen and parameters found for both subpopulations in semen after selection did not correlate with one. This means that information of slow sperm subpopulation kinetics carries important prognostic value of IVF success. Since the current prognosis factors ignore motility parameters of slow sperms, our results show the importance of such an analysis.
Archives of Andrology 07/2009; 51(3):185-93. · 0.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spermatid-specific linker histone H1-like protein (HILS1), transition proteins 1 and 2 (TNP1 and TNP2), and protamines 1 and 2 (PRM1 and PRM2) contribute to considerable dense packing of spermatid chromatin during spermiogenesis. We evaluated the HILS1, TNP1, and TNP2 transcript levels in spermatozoa isolated from normozoospermic and asthenozoospermic men. Human ejaculates from normozoospermic (n = 70) and asthenozoospermic (n = 100) donors were purified by centrifugation through a discontinuous Percoll density gradient. RNA was isolated from spermatozoa according to the Chomczynski and Sacchi method, treated with DNase I and reverse-transcribed into cDNA. Quantitative analysis of HILS1, TNP1, and TNP2 transcripts was performed by real-time quantitative (RQ-PCR) SYBR green I analysis. We found significantly lower levels of HILS1, TNP1, and TNP2 transcripts in spermatozoa from asthenozoospermic men compared to normozoospermic men. Our observations suggest that a reduction in HILS1, TNP1, and TNP2 transcripts may be associated with asthenozoospermia.
Archives of Andrology 07/2009; 53(4):199-205. · 0.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nine different cryoprotectant buffers were tested to measure their protective ability towards main sperm seminological parameters. These were: maintained sperm motility, progressive motility and sperm viability. Out of the nine tested buffers, medium E (TES-Tris without glycerol) and H (glycerol only) showed significantly lower (P < 0.001) values than the rest of the studied buffers in respect to all tested seminological features. The other media did not differ significantly in their cryoprotective abilities to sperm. Richardson's medium (A) preserved sperm viability significantly better (P < 0.001) than the other tested buffers, reaching 63.1% of viable spermatozoa in proportion to the fresh sperm sample before freezing. Three cryoprotectants, A (with egg yolk, no TEST buffer system), D (neither egg yolk nor TEST buffer system), F (TEST-egg yolk buffer system) were further studied for their ability to preserve sperm function in sperm-cervical mucus penetration (Penetrak) and sperm penetration assay (SPA). In our hands, neither supplementation of the buffer with egg yolk nor TEST-egg yolk buffer system promoted sperm capacity in functional tests. A,D,F buffers did not significantly differ among each other in applied functional assays, however, they all diminished (P < 0.001) sperm penetration ratios when compared with fresh sperm samples. Therefore enhancement of sperm capacity to fertilize after equilibration with TEST-egg yolk buffer system should be contested.