Category Archives: Human Reproduction

BACKGROUND

Dysfunction of cellular processes in the testes can lead to infertility, tumourigenesis or other testicular disorders. 14-3-3 proteins are known to play pivotal roles in cellular communication, signal transduction, intracellular trafficking, cell-cycle control, transcription and cytoskeletal structure and have been implicated in several diseases including tumourigenesis. Here we investigated the expression of the 14-3-3 beta isoform in healthy testicular tissues of humans, rats and mice as well as in tissues of Sertoli-cell-only (SCO) syndrome, intratubular germ cell neoplasia (IGCN) and classical seminoma.

METHODS

Samples of healthy and diseased testes from humans, rats and mice were analysed by immunohistochemistry. For PCR, human testis cell lysates were used. Immunoblot analyses of rats and humans healthy testes were performed. Duolink proximity ligation assay (PLA) and co-immunoprecipitation (Co-IP) were carried out to investigate interactions between 14-3-3 beta and vimentin in human, rat and mouse testes.

RESULTS

In healthy testes and SCO syndrome, strong 14-3-3 beta-positive cells could be identified as Sertoli cells. Furthermore, 14-3-3 beta proteins were detected in cells of the peritubular stroma. In samples of IGCN and classical seminoma, the malignant transformed cells stained positive for 14-3-3 beta antigen. Immunoblot analyses revealed the presence of 14-3-3 beta in healthy testicular tissues. 14-3-3 beta mRNA transcripts were detected in cell lysates of healthy human testes. Interaction of 14-3-3 beta with the intermediate filament vimentin was revealed by Duolink PLA and Co-IP. Co-IP experiments identified tubulin as another 14-3-3 beta binding partner.

CONCLUSIONS

Our data suggest that 14-3-3 beta expression is essential for normal spermatogenesis by interacting with vimentin in Sertoli cells. Additionally, 14-3-3 beta expression in malignant transformed cells in IGCN and classical seminoma may lead to tumourigenesis and cell survival.

BACKGROUND

Spermatozoa with large vacuoles (SLV) may have a negative impact on embryo development. The origin of these vacuoles is unknown. We evaluated acrosome and nucleus alterations in isolated SLV, versus unselected spermatozoa.

METHODS

We studied 20 patients with teratozoospermia. Spermatozoa from the native semen sample and spermatozoa presenting a vacuole occupying >13.0% total head area, isolated under high magnification (x6600), were assessed. Confocal and transmission electron microscope evaluations were performed on SLV and native sperm, respectively. Acrosome morphology and DNA fragmentation were analysed using proacrosin immunolabelling (monoclonal antibody 4D4) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Chromatin condensation was evaluated with aniline blue staining. Sperm aneuploidy was assessed using fluorescence in situ hybridization.

RESULTS

SLV represented 38.0 ± 5.10% of motile spermatozoa obtained after gradient density centrifugation. Vacuoles were mainly in the anterior and median sperm head (45.7 ± 2.90 and 46.1 ± 3.00%, respectively). Abnormal acrosomes were increased in SLV compared with unselected spermatozoa (77.8 ± 2.49 versus 70.6 ± 2.62%; P = 0.014). Microscopic observations showed an exclusively nuclear localization of large vacuoles. Complete DNA fragmentation was higher in native spermatozoa (P < 0.0001) than SLV, while chromatin condensation was altered in SLV (P < 0.0001). Aneuploidy and diploidy rates were increased in SLV (P < 0.0001).

CONCLUSIONS

Sperm vacuoles were exclusively nuclear. In our selected teratozoospermic population, aneuploidy and chromatin condensation defects were the main alterations observed in SLV. Based on results from this small sample of spermatozoa, we propose a global impairment of the spermatogenesis process as a common origin of the morphological alterations.

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) Preimplantation Genetic Diagnosis (PGD) Consortium published a set of Guidelines for Best Practice to give information, support and guidance to potential, existing and fledgling PGD programmes (Thornhill AR, De Die-Smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, Moutou C, Robinson MD, Schmutzler AG, Scriven PN et al. ESHRE PGD Consortium best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Hum Reprod 2005;20:35–48.). The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of ESHRE's recent advice on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme; Organization of a PGD centre, fluorescence in situ hybridization-based testing, amplification-based testing and polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Here we have updated the sections that pertain to embryology (including cryopreservation) and biopsy of embryos prior to PGD or PGS. Topics covered in this guideline include uses of embryo biopsy, laboratory issues relating to biopsy, timing of biopsy, biopsy procedure and cryopreserving biopsied embryos.

BACKGROUND

Cryopreservation of ovarian tissue for fertility preservation is based on the ovarian cortex that contains the vast majority of the follicular reserve, while the remaining tissue, the medulla is discarded. The present study describes the development of a gentle method for isolating pre-antral follicles from human ovarian medulla and evaluating its follicular content.

METHODS

Medulla was collected from 40 girls/women aged 3–35 years undergoing cryopreservation of the ovarian cortex. Follicle density was assessed for all patients and pre-antral follicles were isolated from 22 patients. On the basis of the neutral red (NR) staining of follicles and enzymatic digestion with a mixture of Collagenase IV and Liberase Thermolysin Medium, viable pre-antral follicles were isolated.

RESULTS

NR accumulated in follicles resulting in a distinct red staining within the medulla. Follicle density of the medulla varied from 0 to 9824 follicles/gram of medulla and was significantly higher (P< 0.001) in the 3–9-year age group when compared with older groups (10–35 years). Enzymatic digestion combined with follicle identification by NR yielded a high output of isolated and viable pre-antral follicles from medulla, of which, 3607 follicles were collected and classified. The percentage of primordial and growing follicles decreased and increased, respectively, with age (P< 0.0001 and <0.0007).

CONCLUSIONS

Discarded medulla contained a considerable pool of pre-antral follicles, especially in young girls. Our new method allowed the isolation of viable pre-antral follicles from human ovarian medulla and provides a unique opportunity for basic scientific studies and for culture and grafting purposes.

BACKGROUND

PGD has been described in previous cross-sectional and retrospective studies as a stressful experience. No prospective studies of the psychological impact of PGD are currently available.

METHODS

Using a prospective study design, validated measures exploring anxiety and depression were used to assess women using PGD prior to treatment, following embryo transfer, following the pregnancy test result and at 24 weeks of pregnancy. Maternal-fetal attachment was also assessed during pregnancy.

RESULTS

The prospective design revealed the cyclical pathway through PGD for many women, often comprising repeated cycles of ovarian stimulations and IVF and frozen embryo transfers. As predicted, there were significant fluctuations in women's anxiety scores, with increases observed following embryo transfer and pregnancy testing. Women's anxiety scores returned to baseline levels during pregnancy as assessed at 24 weeks gestation. Depression scores did not significantly fluctuate during PGD. Maternal-fetal attachment scores in this sample did not differ from the normative Australian data.

CONCLUSIONS

For some women, the PGD pathway is convoluted and requires multiple IVF cycles and embryo transfers to achieve pregnancy. A subset of women experience significant emotional burden during PGD treatment, and it is these women who require closer attention and support. In this sample, emotional adjustment in pregnancy following PGD appears to be sound.

BACKGROUND

Hepatitis C virus (HCV) carriers are often accepted into the assisted reproduction technique programme of fertility centres. Studies showed that HCV RNA was detected in the follicular fluid of HCV PCR positive females. The objective of this study was to assess the impact of HCV active on the outcome of ICSI.

METHODS

This study was conducted on 40 women who proved to be positive for HCV, using RT–PCR. Two control groups (both n = 40), who were negative for HCV by PCR were also included. The first control group was HCV sero-positive and the second was HCV sero-negative. We compared the three groups regarding the ovarian response to stimulation, embryo quality and pregnancy rates.

RESULTS

The number of failed cycles (lack of ovarian response to stimulation) was higher in HCV RT–PCR positive and sero-positive females than sero-negative controls (P = 0.0001). There were no differences in embryo cleavage or morphology between the study and control groups. The pregnancy rate was significantly reduced in the HCV–PCR-positive group compared with the PCR negative/HCV sero-positive and HCV sero-negative control groups (5, 3 and 48%, respectively; P = 0.001). There was a negative correlation between number of oocytes and viral load (0.419; P = 0.007).

CONCLUSIONS

Our results suggest that HCV infection in females undergoing ICSI has a negative impact on the outcome, and the impact is higher in PCR positive cases: this might be attributed to hormonal disturbance associated with viral liver cirrhosis coinciding with active viral replication.