SCF and c-kit expression profiles in male individuals with normal and impaired spermatogenesis.

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.

The highly conserved NANOS2 protein: testis-specific expression and significance for the human male reproduction.

The highly conserved Nanos gene was found to encode a translational repressor necessary for germ-cell development in lower organisms. The mammalian homologue, Nanos2, was recently found to be expressed in the mouse germ cells. Since its disruption caused infertility exclusively in males, we sought to study the significance of this gene in human male reproduction. Here, we describe for the first time the expression pattern of the NANOS2 gene in human tissues and show that it is testis specific. We found that NANOS2 protein is present in prenatal germ cells and at later stages in spermatogenesis. To elucidate the role of NANOS2 in human germ-line development, we screened this gene for mutations in 214 males with isolated sterility and spermatogenic abnormalities. We identified two heterozygous variants, each in a different oligospermic patient, the second allele being the wild-type. The influence of the first variant, a missense mutation H68Q on the sterility phenotype, was not obvious since it was accompanied by a microdeletion within the AZF region of the Y chromosome. The second variant contained a silent mutation, H109H. Although both mutations were situated within the most conserved RNA-binding domain and were absent in 400 fertile males, it is not obvious that they cause male infertility.

Interleukin-1 superfamily genes expression in normal or impaired human spermatogenesis.

Interleukin-1 (IL-1) is a pleiotropic cytokine that may play a role in contributing to the specific immune environment of mammalian testis and in regulating cell differentiation. We have determined the transcription activity of the IL-1 gene family (using real-time polymerase chain reaction (PCR)) in two main functional testicular compartments (interstitial and intratubular ones), and in tissue homogenates obtained from patients with fertility disorders (spermatogenic arrest and testicular tumors). We observed the prominent expression of gene coding for IL-1 receptor antagonist (IL-1RA) in a purified fraction of gametogenic cells (normal gonad). Caspase-1 (ICE - IL-1beta-converting enzyme) was highly expressed (on mRNA level) in interstitial compartments as well in testicular tumors (immune enhancement?). In addition we found, that the activity of IL-1RA gene decreased along spermatogenic alteration in an inversely related manner with IL-1alpha (from normal gonad through spermatogenic arrest to Sertoli cell only syndrome). Therefore, the quotient value of IL-1alpha/IL-1RA could potentially serve as the diagnostic molecular probe for spermatogenesis assessment. The precise level of mRNA for IL-1-IL-18 cytokines and their receptors, and specifically of the receptor antagonist in immune privileged gonad, could be one of the main factors responsible for maintaining testicular homeostasis, thus enabling generation of the mature spermatozoa.

Polymorphisms of the human PUMILIO2 gene and male sterility.

The highly conserved Pumilio protein plays crucial roles in fertility of many organisms acting as a repressor of translation, and causing infertility when mutated. Although one of two human Pumilio homologs, PUMILIO2 is expressed mainly in the germ line, its role in mammalian germ cell development has not been reported yet. To shed light on the role of PUMILIO2 in development of the human male germ line, we screened this gene for mutations in 137 patients presenting a variety of phenotypes with spermatogenic failure. The first variant, we identified was a single base substitution within intron 15 (IVS15 + 6G > A). This variant was found in three azoospermic males, the second allele being the wild type. However, this variant was also present among fertile males, as frequently as in the patients. Although location of IVS15 + 6G > A substitution in close proximity to the canonical donor splice site GT, indicates that its influence on splicing cannot be excluded, our preliminary cDNA analysis has not revealed evidence of a splicing abnormality of PUMILIO2 pre-mRNA carrying this variant. Nevertheless, this study provides new interesting variant containing a donor splice site variant, which can be relevant for understanding of splicing mechanism of mammalian genes. The second variant, c.774 C > T transversion (Y258Y) in exon 6 was found only in one patient, but an influence on PUMILIO2 function is not obvious. Altogether, this study shows that variation in the PUMILIO2 gene is very low and it seems improbable that mutations of this gene significantly contribute to male infertility in humans.

Quantitative analysis of CCR5 chemokine receptor and cytochrome P450 aromatase transcripts in swim-up spermatozoa isolated from fertile and infertile men.

We determined the CCR5 chemokine receptor and cytochrome P450 aromatase (P450arom) transcript copies number in swim-up sperm isolated from fertile and infertile men. The ejaculates were purified by centrifugation through discontinuous Percoll density gradient and swim-up techniques. RNA was isolated from sperm, treated with DNase I and reverse-transcribed into cDNA. Quantitative analysis of CCR5 and P450arom cDNA were performed by real-time quantitative (RQ-PCR) SYBR Green I analysis. There was a higher content of CCR5 and P450arom transcripts copy number in swim-up sperm of fertile than from infertile donors. The decrease in CCR5 and P450arom transcripts in swim-up sperm may be associated with male infertility.

Effect of sperm subpopulation's kinetics on human fertilization in vitro.

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.

Predictive value of selected sperm parameters for classical in vitro fertilization procedure of oocyte fertilization.

A proportion of fertilized oocytes during classical in vitro fertilization (IVF) procedure was analysed depending on the following factors: number of mature oocytes, seminological criteria such as sperm morphology in raw semen and after its selection in a density gradient (six structural defects of a male gamete were taken into consideration), sperm concentration, motility parameters according to World Health Organization criteria and the functional tests: hypo-osmotic swelling assay and acrosomal reaction induced by calcium ionophore. Evaluation of DNA content in sperm by image cytometry and determination of malonyldialdehydes in seminal plasma were also performed. Seventy-nine semen samples from patients undergoing IVF were assessed. Apart from significant correlations obtained for selected semen parameters and proportion of fertilized eggs, logistic regression analysis showed that the best predictive factors for oocyte fertilization were normal morphology of sperm before and after gradient selection, grade B and C of sperm movement in raw semen, and DNA content after density gradient centrifugation, which all accounted for 76.7% of fertilization predictive value.

[Comparison of the frequency of selected gynecologic operations conducted as laparotomy and laparoscopy during 1993-1997]. / Porównanie czestosci przeprowadzania wybranych typow operacji ginekologicznych na drodze laparotomii i laparoskopii w latach 1993-1997.

UNLABELLED: The aim of the study was to compare frequency of four gynecological operations: myomectomy, tubal surgery, cystectomy and operative management of ectopic pregnancy, performed by laparotomy or laparoscopy, by the same team of surgeons. In the years 1994-1997 in Division of Reproduction Poznan University Medical School 647 cystectomies, 208 myomectomies, 68 tuboplasties and 50 surgical treatments of ectopic pregnancy were done. Among 973 operations--684 (70.3%) were performed by laparoscopy. There was a gradual tendency in increasing endoscopic procedures. Comparing the year 1994 and 1997 percentage of operations performed by laparoscopy significantly changed: In tuboplasty from 83% to 95%, cystectomy from 35.9% to 80.3%, ectopic pregnancy from 61.5% to 91.7% and myomectomy from 52.7% to 61.5%. Patient hospital stay decreased significantly after laparoscopic procedures (from 5.1 days to 3.25 days). During the study period open surgery followed laparoscopy only in 8 cases (1.1%) because of complications or technical difficulties. CONCLUSION: 1. Operative laparoscopy is a safe and effective procedure, in many cases replacing open surgery. 2. Shortening of hospital stay and recovery period after laparoscopy is one of the main advantages of this method of treatment.

Idiopathic infertility in married couples in the light of cytogenetic analysis and sperm penetration assay.

We have selected 47 couples with unexplained infertility in order to analyse a possible link between sperm dysfunction studied in males in in vitro conditions and karyotype analysis of somatic cells. In order to identify so called "idiopathically infertile" couples we had to exclude any change in reproductive organs in both partners or in spermiogram which would qualify any of spouses into known category of infertility. We have revealed chromosome aberrations (translocations and marker chromosomes) in 19% of infertile males and in 6% of infertile females. Idiopathically infertile males had an overall decreased ability of sperm function (measured by proportion of penetrated hamster oocytes by human sperm) in comparison to fertile controls, however, still well placed within physiological range of values. Only sperm from a patient with identified translocation was clearly below the normal level of penetration (20% of penetrated oocytes), however, also the patients with revealed chromosome variant polymorphisms presented statistically lower values of penetration in comparison to fertile controls (39% vs 57%, p<0.05). On the contrary, patients with marker chromosomes did not exhibit affected sperm function. It can be speculated that only particular chromosome aberration in group of idiopathically infertile males may affect sperm functional capability (measured in vitro), however, the intragonadal genetic analysis has to be recommended in order to confirm such a causative link.

[Importance of cytogenetic analysis in patients with azoospermia or severe oligozoospermia undergoing in vitro fertilization]. / Znaczenie badan cytogenetycznych u pacjentów z azoospermia lub ciezka postacia oligozoospermii, korzystajacych z zaplodnienia in vitro.

The karyotypic analysis was performed to assess the importance of genetic factor in male infertility. For that purpose, chromosomal analysis in blood lymphocytes was performed in 28 males, candidates for ICSI with azoospermia or severe oligozoospermia and in their spouses. Although chromosomal aberrations were identified in as many as 11 couples, (in 6 couples aberrations were identified in male, in 4 other couples in female partner, whereas in 1 one couple they were detected in both partners) their risk for potential offspring is unequal. Balanced autosomal aberrations detected in two males (7%) constitute a high risk since they can cause not only infertility but also severe somatic abnormalities if transferred as the unbalanced ones to the next generation. The remaining 9 chromosomal aberrations identified in this study were present in mosaic additional cell lines with low representation. In 8 of them sex chromosomes and in 1 an autosom were involved. Although these mosaic chromosomal aberrations can lower efficiency of in vitro fertilisation, the probability that they can be transferred to the next generation causing somatic abnormalities is not high. This study indicates that in case of azoospermia or severe oligozoospermia, the karyotypic analysis should be performed in both partners prior to in vitro fertilisation.

[Laparoscopic myomectomy]. / Laparoskopowe leczenie miesniaków macicy.

OBJECTIVES: The aim of our study was clinical analysis of the factors influencing on laparoscopic myomectomy. STUDY DESIGN: Retrospective analysis of the operative protocols. MATERIAL AND METHODS: Two hundred nineteen women had laparoscopy because of unexplained infertility (n = 109) unexplained infertility and myomas (n = 41), myomas (n = 36), endometriosis suspicion (n = 20) ovarian cyst (n = 9) or pelvic pain syndrome (n = 4). RESULTS: Among 299 myomas 186 were extirpated during laparoscopy. In 39 cases suturing of the myometrium was necessary. Electrocautery was performed in 27 cases and laser-vaporisation in 8. In 28 women the operation was postponed because of small myomas and mainly poor operative technique (beginning of the learning curve). In two of them second laparoscopy was performed after GnRH therapy. An analysis of the factors which enable laparoscopic myomectomy was performed. The most important factors are: size and number of the myomas, localization in the myometrium, experienced hands and operative room equipment. CONCLUSIONS: Uterine myomas are one of the indications to operative laparoscopy. Meticulous analysis of the operative conditions as well as the assessment of the team experience should always precede laparoscopy.

[The influence of FSH stimulation on the results of intrauterine insemination]. / Wplyw stymulacji cyklu za pomoca fsh na wyniki inseminacji nasieniem homologicznym.

OBJECTIVE: To compare the efficacy of IUI husband in natural versus FSH stimulated cycles. DESIGN: Prospective, controlled study. MATERIALS AND METHODS: IUI were performed in 57 infertile couples with natural cycles, and in 16 under FSH and GnRH stimulation (Short protocol). In stimulated patients also hCG and hydrogesteron were given. Indication in both groups was idiopathic infertility. Duration of infertility and the age were comparable. Semen preparation and ovarian monitoring were the same in 2 groups. RESULTS: Three pregnancies in 57 natural IUI cycles (5.3%) and 5 out of 16 cycles in stimulated women (31.2% per cycle-with one triple pregnancy). CONCLUSION: In couples with idiopathic infertility FSH stimulation significantly increases rate of pregnancy and multiple gestation.

Severe antioxidase deficiency in human semen samples with pathological spermiogram parameters.

Spermatozoa of 103 ejaculates from infertile patients and fertile healthy individuals were separated from seminal plasma and purified on Percoll gradient to determine the activities of superoxide dismutase (SOD) and catalase (CAT) in seminal plasma as well as in spermatozoal supernatants after hypotonic disintegration of the sperm plasma membrane. Out of collected specimens, a subgroup of ejaculates from 40 individuals was examined whose female partners had developed malignant processes in the cervix uteri (oncological subgroup). All sperm samples were classified into normal and pathological semen samples according to WHO criteria. While no significant differences of SOD levels were detected in seminal plasma of patients with primary infertility, a catalase deficiency seemed to be associated with combined sperm pathology-oligoasthenoteratozoospermia (OAT). Liberated concentrations of both SOD and catalase were diminished by 10-70% in the oncological subgroup compared to normozoospermia. In four OAT samples obtained from infertile males of the oncological subgroup, total depletion from both antioxidases was observed. A lack of sufficient antioxidase protection in cases of severe sperm pathology (OAT) may also lead to cervical dysplasia.

[Bacterial flora of genital organs in women treated for infertility]. / Flora bakteryjna narzadów plciowych kobiet leczonych z powodu nieplodnosci.

Bacterial flora in 72 women suffering from infertility was assessed. The medium age of women was 28.9 years (22-37) and the duration of infertility-5.5 years (2-15). Cultures were taken during laparoscopy (65) or microsurgery (7) from vagina, cervical canal and the fallopian tubes. Specific cultures for anaerobic and aerobic bacteria, N. gonorrhoeae, C. Trachomatis and Mycoplasma hominis were taken. In the specimens from fallopian tubes 21 species of bacteria were cultured. The most often coagulase-negative staphylococci (54.3%) as well as aerobic and anaerobic Gram-positive bacilli were found (23.9%). In conclusion we think that the proper preparation of patients in the preoperative period decreases significantly the number of isolated species of bacteria. Also the profile of cultured species differs from that found in women from western populations, especially from United States and Sweden.

Effect of reactive oxygen species and the activity of antioxidant systems on human semen; association with male infertility.

A range of compounds with a role in oxidative stress were measured in ejaculates from 40 normozoospermic individuals and 93 infertile males. Ejaculates were classified according to WHO criteria. Seminal plasma and the sperm cell fraction were assessed separately for superoxide dismutase (SOD), catalase, xanthine oxidase, capability for singlet oxygen trapping and content of thiobarbituric acid-reactive substances (TBARS). Pathological cases defined as oligozoospermia, asthenozoospermia, or teratozoospermia revealed different backgrounds of oxidative stress as reflected by different levels of tested substances in every type of sperm pathology. In the majority of abnormal ejaculates, a significant increase in intracellular activity of SOD, decreased intracellular levels of catalase, elevated levels of xanthine oxidase and TBARS, and severely impaired singlet oxygen trapping were observed when compared to normozoospermic ejaculates. Interrelationships between SOD and TBARS, and between xanthine oxidase and catalase, appeared to be of key importance when analysed separately in seminal plasma and in spermatozoa or in a combination of both. Elevated xanthine oxidase levels and low capacity for singlet oxygen trapping are statistically significant factors for the evaluation of male infertility which can develop as a result of persistent oxidative stress.

[Usefulness of counting leukocytes and round cells in determination of bacterial infection of semen in infertile men]. / Przydatnosc oznaczania leukocytów i komórek okraglych w okresleniu zakazenia bakteryjnego nasienia nieplodnych mezczyzn.

The authors measured efficiency of the determination of bacterial infection from the semen of infertile men. They compared the correlation of counts of bacteria with white blood cells (peroxidase method) and round cells in ejaculate. Seminal white blood cells counts (more than 1 x 10(5)/ml) correlated well with bacteriospermia (more than 1000 cells/ml), R = 0.4741, p = 0.0007. Determination of round cells in semen has no clinical value in prediction of seminal infection, R = 0.0153, p = 0.6022.

Oxidative stress and male infertility.

We have studied the activity of substances (superoxide dismutase [SOD], catalase [Cat], malonaldehyde, xanthine oxidase [XO], nitric oxide [NOx]) participating in oxidative stress. Seminal plasma samples of 147 ejaculates obtained from normal and from infertile males were examined. Activities of SOD, Cat, and XO were measured chemiluminometrically while malonaldehydes and NOx were measured by spectrophotometer in seminal plasma samples. Ejaculates were previously characterized according to World Health Organization andrological criteria (sperm number, motility, and morphology). Procedures were performed in a university laboratory. Statistically significant changes (in comparison to normozoospermic samples) were noted in activities of SOD, XO, and malonaldehyde levels. The SOD activity exceeded values obtained for normozoospermic samples only in oligozoospermia. Otherwise low SOD levels in analyzed infertile subgroups inversely related to elevated malonaldehydes. Because diminished activity of SOD in seminal plasma was associated with increased levels of malonaldehydes and XO, we could postulate some significance of these monitored substances in evaluation of the cause of male infertility.

Seminal plasma can be a predictive factor for male infertility.

Seminal plasma from ejaculates of 10 healthy, fertile volunteers and 63 infertile males was analysed for superoxide dismutase (SOD) and xanthine oxidase (XO) activities using a chemiluminometer. There was not statistically significant difference in the activity of either enzyme between control and infertile populations (113 +/- 74 IU/ml for SOD and 1.17 +/- 0.52 IU/ml for XO) in samples from normozoospermic ejaculates. Sperm progressive motility was positively correlated with SOD activity in seminal plasma of corresponding ejaculates (P < 0.05) and negatively with XO activity (P < 0.001). An 'oxido-sensitive' index was defined as the SOD/XO ratio and was found to be inversely related to sperm progressive motility samples (P < 0.01). Analysing this index among all tested samples of semen including those with pathological spermiograms, as well as normospermic (N) samples we found statistically significant (elevated) differences in oligoasthenoteratospermia (OAT) in comparison with N (P <0.05); OAT samples were also significantly different from oligospermic (O) and oligoteratospermic (OT) samples (P < 0.05). This suggests that the 'oxido-sensitive' index of seminal plasma may be a simple diagnostic factor, useful in the determination of male infertility.

Evaluation of sperm cryoprotective media in respect to fertilizing capacity tested in vitro.

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.