Attila Jakab

Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects

The testis-expressed chaperone protein, HspA2 (previously creatine kinase M isoform) was established as a measure of human sperm cellular maturity, function and fertility. The presence of HspA2 in the synaptonemal complex is likely to link low HspA2 expression and increased frequency of chromosomal aneuploidies in arrested-maturity spermatozoa. A relationship also exists between HspA2 expression in elongating spermatids and the associated spermatogenetic events, including plasma membrane remodelling and the formation of zona pellucida and hyaluronic acid (HA) binding sites. The HA receptor of mature spermatozoa, when coupled with HA-coated slides and/or Petri dishes, allows visual observation of sperm-HA binding, providing a basis for sperm maturity testing, a major improvement in semen evaluation, and selection of mature spermatozoa for intracytoplasmic sperm injection (ICSI). Thus, in HA-selected spermatozoa the frequency of chromosomal disomy and diploidy is reduced 4- to 6-fold compared with semen sperm fractions. This reduction is similar to the increase in numerical chromosomal aberrations in ICSI children. Combined studies of sperm shape and chromosome probes demonstrated that sperm morphology does not aid selection of haploid spermatozoa. The HA-mediated sperm selection is a novel and efficient technique that may alleviate potential problems related to ICSI fertilization with visually selected spermatozoa.

Biochemical markers of sperm function: male fertility and sperm selection for ICSI.

The expression of a 70 kDa chaperone protein, HspA2 (formerly called CK-M), has been identified in mature human spermatozoa. The central role of HspA2 has been established, as the expression level of this protein is related to sperm cellular maturity, DNA integrity, chromatin maturity, chromosomal aneuploidy frequency and sperm function, including fertilizing potential. The spermiogenetic events of cytoplasmic extrusion and remodelling of the plasma membrane, which facilitate the formation of zona pellucida binding site(s) in human spermatozoa, are related. Finally, the presence of the hyaluronic acid (HA) receptor on the plasma membrane of mature sperm coupled with the HA-coated slide sperm-binding assay, facilitates the testing of infertile men and the selection of single mature spermatozoa for ICSI. Because mature spermatozoa have no residual cytoplasm, the HA-bound sperm fraction is also enriched in spermatozoa that are normal by the Kruger strict morphology method.

Hyaluronic acid binding ability of human sperm reflects cellular maturity and fertilizing potential : selection of sperm for intracytoplasmic sperm injection

Purpose of review The current concepts of sperm biochemical markers and the central role of the HspA2 chaperone protein, a measure of sperm cellular maturity and fertilizing potential, are reviewed. Recent findings Because HspA2 is a component of the synaptonemal complex, low HspA2 levels and increased frequency of chromosomal aneuploidies are related in diminished maturity sperm. We also suggest a relationship between HspA2 expression in elongating spermatids and events of late spermiogenesis, such as cytoplasmic extrusion and plasma membrane remodeling that aid the formation of the zona pellucida binding and hyaluronic acid binding sites. The presence of hyaluronic acid receptor on the plasma membrane of mature sperm, coupled with hyaluronic acid coated glass or plastic surfaces, facilitates testing of sperm function and selection of single mature sperm for intracytoplasmic sperm injection. The frequencies of sperm with chromosomal disomy are reduced approximately fourfold to fivefold in hyaluronic acid selected sperm compared with semen sperm, comparable to the increase in such abnormalities in intracytoplasmic sperm injection offspring. Hyaluronic acid binding also excludes immature sperm with cytoplasmic extrusion, persistent histones, and DNA chain breaks. Summary Hyaluronic acid mediated sperm selection is a novel technique that is comparable to sperm zona pellucida binding. Hyaluronic acid selected sperm will also alleviate the risks related to intracytoplasmic sperm injection fertilization with sperm of diminished maturity that currently cause worldwide concern.

Correlation study between sperm concentration, hyaluronic acid-binding capacity and sperm aneuploidy in Hungarian patients.

Infertile men with low sperm concentration and/or less motile spermatozoa have an increased risk of producing aneuploid spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding may reduce genetic risks such as chromosomal rearrangements and numerical aberrations. Fluorescence in-situ hybridization (FISH) has been used to evaluate the presence of aneuploidies. This study examined spermatozoa of 10 oligozoospermic, 9 asthenozoospermic, 9 oligoasthenozoospermic and 17 normozoospermic men by HA binding and FISH. Mean percentage of HA-bound spermatozoa in the normozoospermic group was 81%, which was significantly higher than in the oligozoospermic (P<0.001), asthenozoospermic (P<0.001) and oligoasthenozoospermic (P<0.001) groups. Disomy of sex chromosomes (P=0.014) and chromosome 17 (P=0.0019), diploidy (P=0.03) and estimated numerical chromosome aberrations (P=0.004) were significantly higher in the oligoasthenozoospermic group compared with the other groups. There were statistically significant relationships (P<0.001) between sperm concentration and HA binding (r=0.658), between sperm concentration and estimated numerical chromosome aberrations (r=-0.668) and between HA binding and estimated numerical chromosome aberrations (r=-0.682). HA binding and aneuploidy studies of spermatozoa in individual cases allow prediction of reproductive prognosis and provision of appropriate genetic counselling. Infertile men with normal karyotypes and low sperm concentrations and/or less motile spermatozoa have significantly increased risks of producing aneuploid (diminished mature) spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding, based on a binding between sperm receptors for zona pellucida and HA, may reduce the potential genetic risks such as chromosomal rearrangements and numerical aberrations. In the present study we examined sperm samples of 45 men with different sperm parameters by HA-binding assay and fluorescence in-situ hybridization (FISH). Mean percentage of HA-bound spermatozoa in the normozoospermic group was significantly higher than the oligozoospermic, the asthenozoospermic and the oligoasthenozoospermic groups. Using FISH, disomy of sex chromosomes and chromosome 17, diploidy and estimated numerical chromosome aberration frequencies were significantly higher in the oligoasthenozoospermic group compared with the three other groups. A significant positive correlation was found between the sperm concentration and the HA-binding capacity, and significant negative correlations between the sperm concentration and the estimated numerical chromosomes aberrations as well as between the HA-binding ability and the estimated numerical chromosome aberrations were identified. We conclude that HA-binding assay and sperm aneuploidy study using FISH may help to predict the reproductive ability of selected infertile male patients and to provide appropriate genetic counselling.

Is sperm hyaluronic acid binding ability predictive for clinical success of intracytoplasmic sperm injection: PICSI vs. ICSI?

Abstract Although intracytoplasmic sperm injection (ICSI) is now a widely-used technique, it is still of interest to improve our knowledge as to which is the best spermatozoon to be selected for ICSI. Infertile men have increased risks of producing aneuploid spermatozoa. Using hyaluronic acid (HA)-binding sperm selection may reduce the genetic risks such as chromosomal aberrations of offspring. In the present study we examined the clinical success of ICSI with HA-selected sperm (‘physiologic’ ICSI, PICSI) compared to conventional ICSI, as well as the necessity to differentiate patients according to the initial HA-binding assay result (HBA score) and whether the sperm concentration or HBA score can provide additional information. We observed a significantly higher fertilization rate (FR) of the PICSI group with >60% HBA, implantation rate (IR) of the PICSI group with ≤60% HBA, and clinical pregnanacy rate (CPR) in every PICSI group compared to the ICSI groups (p < 0.01). We also observed a significantly higher life birth rate (LBR) in the PICSI group with ≤60% HBA compared to ICSI patients with ≤60% HBA (p < 0.001). The pregnancy loss rate (PLR) was significanly lower in PICSI patients compared to the ICSI group (p < 0.0001). The FR, IR, CPR, and LBR of the PICSI group with <50% HBA were significantly higher and the PLR was lower than in the ICSI group with <50% HBA (p < 0.01). A statistically significant correlation was found between the sperm concentration and the HA-binding capacity (r = 0.62, p < 0.001). We found a closer relationship between HBA score and FR (r = 0.53, NS) than between sperm concentration and FR (r = 0.14, NS). HBA could be considered for sperm selection prior to ICSI because of its success and apparant ability to reduce genetic complications. However, this must be extended to a larger study.

Meiotic segregation study of a novel t(3;6)(q21;q23) in an infertile man using fluorescence in situ hybridization (FISH)

Male carriers with balanced reciprocal translocations can produce a variable proportion of unbalanced gametes resulting in reproductive failures. The presence of a structural rearrangement may induce an interchromosomal effect. This is characterized by abnormal bivalents not involved in the reorganization thereby yielding non-disjunction, which would present as aneuploid spermatozoa for these chromosomes. In the present case report segregation analysis of the sperm and investigation of interchromosomal effect were carried out using cytogenetic and fluorescence in situ hybridization (FISH) analysis on blood lymphocytes. The karyotype of the patient was 46,XY,t(3;6)(q21;q23). During sperm segregation analysis a total of 2,002 sperms were evaluated, of which 46.8% showed normal/balanced (alternate segregation mode) and 53.2% of sperm showed an abnormal signal pattern. A significant difference in the frequency of the estimated number of chromosome anomalies was observed in the translocation carrier when compared to the normozoospermic group (P < 0.0001) and the oligozoospermic group (P < 0.0001). Meiotic segregation analysis of sperm together with aneuploidy assessment for X, Y, and 17 chromosomes using FISH allows for the determination of a reproductive prognosis in male balanced translocation carriers and can be used for appropriate genetic counseling.

Adequacy of hyaluronan binding assay and a new fertility index derived from it for measuring of male fertility potential and the efficacy of supplement therapy

The aim of the study was to statistically prove that the HBA® test is an efficient practical method for andrologists to determine the fertility potential as well as to measure the efficiency of oral supplement therapy in case of male infertility. In the study, 175 patients were involved and it also included the follow‐up studies of 39 patients after supplement therapy. Completing the ‘classic’ spermatological parameters with the results of HBA® test, the authors have also determined a new fertility index to be used for practical rating of the measure of fertility potential. After the supplement therapy, both sperm density and hyaluronan binding capacity increased significantly. The authors are convinced that the HBA® analysis is an objective, standardisable test, which provides a better approach to fertility potential. This analysis enables us to detect spermatozoa that were previously misjudged as normal by morphological assay and also makes the efficiency of the therapy more measurable.

[Influence of malignant tumors occurring in the reproductive age on spermiogenesis: studies on patients with testicular tumor and lymphoma].

INTRODUCTION The application of chemo- and radiotherapy results in good survival prognosis for young men with malignant tumors, but long-term gonadoxic effect has to be considered. In addition, malignant disease itself has a negative impact on spermiogenesis. AIM The aim of the authors was to examine the spermiogenetic effect of the most common tumors occurring in the reproductive age in men: testicular cancer, Hodgkin disease and non-Hodgkin disease. METHOD Spermiogram of men with testicular cancer (N = 68), Hodgkin disease (N = 37) and non-Hodgkin disease (N = 14) who were referred for sperm cryopreservation were analysed in the Reproductive Andrology Laboratory of the authors. RESULTS Azoospermia was found in 11.8% of all patients (N = 119), while 58.8% of the patients had oligozoospermia even before the treatment. Sperm concentration of men with testicular cancer was significantly lower than those with lymphomas (32.8 M/mL vs. 24.9 M/mL, p = 0.03). There was no difference in sperm concentration between the Hodgkin and non-Hodgkin lymphoma groups. CONCLUSIONS Spermiogenetic defect is more pronounced in men with testicular cancer than those with lymphomas. Cryopreservation before treatment for fertility preservation should be offered for all reproductive aged men with malignant disease, especially for those with testicular cancer.

Body image and quality of life in women with polycystic ovary syndrome

INTRODUCTION Polycystic ovary syndrome is the most common endocrine disease in women. Psychic consequences are significant; patients have difficulties to be pregnant and the disease disturbs the quality of life. Due to complications associated with polycystic ovary syndrome, studies on psychological state and disease perception of patients appear to be important. AIM The aim of the authors was to assess how the disease influences quality of life and changes in body image. METHOD The authors used the Illness Intrusiveness Ratings Scale, Health-Related-Quality-of-Life Questionnaire for Women with Polycystic Ovary Syndrome and their own body image questionnaire. 121 women took part in the study. RESULTS There was a negative correlation between illness intrusiveness and quality of life (r = -0.353) and a positive correlation between body image and quality of life (r = 0.614). A significant difference was observed in body satisfaction between patients who had visible body changes and those who had metabolic disturbances (p<0.05). Overweight patients who lost weight with lifestyle changes had significantly better quality of life (p<0.05). CONCLUSIONS These results appear to be convincing in that in addition to gynecological care, psychotherapy could be offered to patients with polycystic ovary syndrome.

Plasminogen Activator Inhibitor Type 2 in Human Tears and Blood during Pregnancy

Purpose: During pregnancy, systemic blood levels of plasminogen activator inhibitors (PAIs), estradiol (E2) and progesterone (P4) are expected to rise with gestational age. This work examines whether a corresponding rise in PAIs occurs in tears. Methods: Tear and blood samples were collected opportunistically from women during pregnancy: PAI-2 and PAI-1 levels were measured using enzyme-linked immunosorbent assay (ELISA) tests. Blood levels of E2 and P4 were measured using chemiluminescence immunoassays. Results: Levels of PAI-2 from tears of pregnant women did not increase and did not correlate with gestation. Levels of PAI-2, E2 and P4 in blood significantly increased over gestation. Tear PAI-2 levels did not correlate with blood levels of PAI-2, E2 or P4. Although blood levels of PAI-1 increased with gestation, tear levels of PAI-1 were below the ELISA detection limit, similar to the situation for non-pregnant subjects. Conclusions: Tear PAI-2 levels are dissociated from the progression of pregnancy and the concomitant elevated systemic blood PAI-2, E2 and P4 levels, indicating the possibility of local control of proteolysis in the eye during pregnancy in the absence of wounding or surgery on the eye.