Sinan Ozkavukcu

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.

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.

Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa

PurposeCryopreservation of sperm is a widely used technique to maintain and protect the fertility in various occasions such as infertility and malignancy treatments. This study aims to reveal the effects of freezing and thawing on human spermatozoa.Materials and methodsTo evaluate the effects of freeze–thawing, semen samples were evaluated by light microscopy by means of morphology, motility and viability, by scanning and transmission electron microscopy for detailed ultrastructural changes.ResultsAfter cryopreservation, a significant decrease in spermatozoa viability was observed (p < 0.01). Group a, b and c motility according to World Health Organization criteria decreased considerably (p < 0.05, p < 0.01, p < 0.05, respectively), whereas there was a substantial increase in group d motility. A strong correlation between rise in number of immotile spermatozoa and decrease in viability was also noted (r = −0.848, p < 0.01). Post-thaw light microscopic studies revealed a considerable decrease in rate of normal spermatozoa (p < 0.05). A considerable decline in the rate of normal sperm was also observed by TEM (p < 0.05). Statistically, acrosomal changes and subacrosomal swelling were found to be significantly increased (both p < 0.05), where the latter appears to be a novel finding in literature.ConclusionCryopreservation has deleterious effects on spermatozoa, especially on plasmalemma, acrosomes and tails. Electron microscopy is the ultimate modality to investigate spermatogenic cells.

Selectivity of hyaluronic acid binding for spermatozoa with normal Tygerberg strict morphology

During spermiogenesis, a plasma membrane remodelling step facilitates formation of sperm zona pellucida and hyaluronic acid (HA) binding sites. Enrichment of Tygerberg normal spermatozoa in HA-bound versus semen sperm fractions was postulated. Semen was placed on the uncoated A side and HA-coated B side of a semen chamber. After 15 min, the HA binding score (proportion of HA-bound motile spermatozoa) was assessed on the B side, and unbound spermatozoa were removed by gentle rinsing. Following Diff-Quick staining, sperm morphology of A and B sides was evaluated by three blinded investigators at Yale and Tygerberg. The proportion of Tygerberg normal spermatozoa was higher in HA-bound versus semen spermatozoa (n = 63 subjects) with a 3.04-fold improvement (95% confidence limits: 1.9-4.7) in 37 teratozoospermic men, comparable with a 4.2-fold enrichment in zona pellucida-bound spermatozoa reported earlier by the Tygerberg group. The morphology scores of three investigators were different but related, indicating that the variations reflect individual-to-individual differences in the perception of shape normality. The selection power of HA and zona pellucida for normal spermatozoa are similar. The sperm biomarkers of creatine phosphokinase (reflecting retained cytoplasm in arrested maturity spermatozoa) and chaperone protein HspA2 (heat shock protein) were proportional with sperm HA binding. As HA binding reflects sperm maturity and function, the combination of Tygerberg morphology and HA binding is likely to improve male infertility management.

Does combining magnetic-activated cell sorting with density gradient or swim-up improve sperm selection?

PurposeThe present study aimed to evaluate whether combining the magnetic-activated cell sorting (MACS) with density-gradient (DG) or swim-up (SU) sperm separation techniques can improve sperm selection to obtain higher quality spermatozoa.MethodsTwo commonly used sperm selection techniques, SU and DG, were compared to MACS combined with either SU or DG. Spermatozoa obtained from normozoospermic (n = 10) and oligozoospermic (n = 10) cases were grouped as SU, DG, SU+MACS, and DG+MACS followed by the analysis of sperm morphology, motility, DNA integrity, and the levels of Izumo-1 and PLCZ proteins.ResultsAlthough spermatozoa obtained by SU or DG when combined with MACS have improved aspects when compared to SU or DG alone, results did not reach a statistically significant level. Moreover, separation with MACS caused a significant loss in the numbers of total and rapid progressive spermatozoa.ConclusionsConsidering the cost/benefit ratio, MACS application together with traditional techniques may only be preferred in certain cases having higher concentrations of spermatozoa, but it does not seem to be an ideal and practical sperm selection technique for routine use.

A laboratory modification to testicular sperm preparation technique improves spermatogenic cell yield.

Testicular sperm extraction is a common procedure used to find spermatogenic cells in men with nonobstructive azoospermia. The laboratory processing of biopsied testicular tissues needs to be performed meticulously to acquire a high yield of cells. In this study, the effectiveness of mincing the tissues after testicular biopsy was assessed using histological evaluation, as was the possible adverse effect of residual tissue on the migration of spermatogenic cells during density gradient centrifugation. Our results indicate that testicular residual tissue, when laid on the density gradient medium along with the sperm wash, hinders the spermatogenic cells’ forming a pellet during centrifugation, and therefore impairs the intracytoplasmic sperm injection procedure. Whereas the mean number of recovered cells from the sperm wash medium (SWM) with residual tissue is 39.435 ± 24.849, it was notably higher (60.189 ± 28.214 cells) in the SWM without minced tissues. The remaining tissue contained no functional seminiferous tubules or spermatogenic cells in histological sections. In conclusion, the remaining residual tissue after mincing biopsied testicular tissue does not add any functional or cellular contribution to spermatogenic cell retrieval; in fact, it may block the cellular elements in the accompanying cell suspension from migrating through the gradient layers to form a pellet during centrifugation and cause loss of spermatogenic cells.

Outcomes after early or midfollicular phase LH supplementation in previous inadequate responders.

Second cycle outcomes of 75 patients who had previous inadequate ovarian response with recombinant FSH (rFSH)-only ovarian stimulation during gonadotrophin-releasing hormone analogue (GnRHa) down-regulated cycles were evaluated retrospectively. In these second cycles, both rFSH and human menopausal gonadotrophin (HMG) in GnRHa long down-regulation were given to all patients, HMG initiated either on day 1 (group A, n=37) or day 5-6 of the ovarian stimulation (group B, n=38). Total HMG dose was higher (1198+/-514 IU versus 726+/-469 IU; P<0.001), cumulative rFSH consumption was lower (1823+/-804 IU versus 2863+/-1393 IU; P=0.001) and duration of stimulation was shorter (8.94+/-1.15 days versus 10.37+/-1.80 days; P<0.001) in group A than in group B. No significant differences were found regarding fertilization, implantation or pregnancy rates and embryo quality between the groups. Further analysis by supplementary HMG dose (75 IU versus 150 IU) revealed that total gonadotrophin and HMG consumption was lower in 75 IU-supplemented subgroups. Notably, pregnancy rate was higher in patients where 75 IU HMG was supplemented on day 5-6 of ovarian stimulation, which deserves further evaluation.

Rare presentation of ectopic pregnancy following IVF-ET: live twin gestation in the same fallopian tube

We report a case of a twin ectopic pregnancy (EP) after in vitro fertilization and embryo transfer (IVF-ET). A 24-year-old nulligravida presented with lower abdominal pain and vaginal bleeding 4 weeks after embryo transfer. Serum β-HCG levels were 40 IU/mL, 90 IU/mL, and 1970 IU/mL on ET days 12, 14, and 23, respectively. Ultrasound examination revealed two ectopic gestational sacs with fetal heart beats in the left adnexa, without evidence of intrauterine pregnancy. At laparoscopy, one isthmic and another ampullary sac were detected in the left tube and left salpingectomy was performed. The patient was discharged healthy on postoperative day 2. Albeit extremely rare, ectopic pregnancies with abnormal presentation can be encountered following IVF-ET. Single embryo transfer may be advised to protect from ectopic pregnancies after IVF-ET.

In Vivo acrylamide exposure may cause severe toxicity to mouse oocytes through its metabolite glycidamide

High acrylamide (ACR) content in heat-processed carbohydrate-rich foods, as well as roasted products such as coffee, almonds etc., has been found to be as a risk factor for carcinogenicity and genotoxicity by The World Health Organization. Glycidamide (GLY), the epoxide metabolite of ACR, is processed by the cytochrome P-450 enzyme system and has also been found to be a genotoxic agent. The aim of this study was to determine whether ACR and/or GLY have any detrimental effect on the meiotic cell division of oocytes. For this purpose, germinal vesicle-stage mouse oocytes were treated with 0, 100, 500, or 1000 μM ACR or 0, 25, or 250 μM GLY in vitro. In vivo experiments were performed after an intraperitoneal injection of 25 mg/kg/day ACR of female BALB/c mice for 7 days. The majority of in vitro ACR-treated oocytes reached the metaphase-II stage following 18 hours of incubation, which was not significantly different from the control group. Maturation of the oocytes derived from in vivo ACR-treated mice was impaired significantly. Oocytes, reaching the M-II stage in the in vivo ACR-treated group, were characterized by a decrease in meiotic spindle mass and an increase in chromosomal disruption. In vitro GLY treatment resulted in the degeneration of all oocytes, indicating that ACR toxicity on female germ cells may occur through its metabolite, GLY. Thus, ACR exposure must be considered, together with its metabolite GLY, when female fertility is concerned.

A differential cytokine expression profile before and after rFSH treatment in Sertoli cell cultures of men with nonobstructive azoospermia

To evaluate the effects of follicle‐stimulating hormone (FSH) treatment on cytokine gene expression in cultured Sertoli cells from men with nonobstructive azoospermia, a total of 15 azoospermic men diagnosed as obstructive azoospermia (OA) (n = 5) and nonobstructive azoospermia (NOA) (n = 10) were included in the study. NOA patients were split into two further subgroups: nFSH and hFSH serum FSH levels. Expression of cytokine gene panel (88 genes), FSHR and ABP was evaluated by real‐time PCR array analysis. FSHR protein level was measured by the Western blot. In primary cultures of Sertoli cells, seven genes were found to be increased and 13 were decreased in NOA group, when compared to OA (p < .05). When rFSH was introduced into the culture media, expression of 12 genes in the NOA group restored a comparable level to those of the control OA group. Sertoli cells in all groups responded rFSH administration with increased expression of ABP. Our results suggest that FSH treatment may have positive effects on Sertoli cells of nonobstructive azoospermic patients via changing the expression levels of certain genes and restoring their levels in normal Sertoli cell population. Some cytokine levels can be considered as a potential candidate for detecting NOA patients. ABP is a good marker for cell viability and functionality in primary Sertoli cell culture.