Comparison of sperm preparation methods to improve the recovery of mature spermatozoa in sub-fertile males.

The integrity of chromatin in the spermatozoon is essential for reproductive outcome. The aim of this study was to evaluate the most effective and cost-effective method to reduce the percentage of spermatozoa with defects in chromatin decondensation for use in assisted reproductive technologies (ART) procedures. Sperm samples from 15 sub-fertile males were examined at CFA Naples to determine the sperm decondensation index (SDI), using the aniline blue test, before and after preparation, comparing density gradients with two different swim-up approaches. All three techniques led to a reduction in decondensed spermatozoa with no statistical difference (P > 0.05) between the control and the treated sperm. In contrast, we found a highly significant decrease in SDI (P < 0.01) after the two swim-up methods in all the samples, confirming the efficacy of these methods in lowering the percentage of chromatin compaction damage. There was no statistical difference between the two swim-up methods, however swim-up from the pellet led to improved count, motility and the percentage of normal condensed spermatozoa. We suggest that swim-up from the pellet be used in ART on sub-fertile males, both to reduce cell stress by multiple centrifugation and improve the recovery rate of mature spermatozoa.

The Harderian gland: Endocrine function and hormonal control.

The Harderian gland (HG) is an exocrine gland located within the eye socket in a variety of tetrapods. During the 1980s and 1990s the HG elicited great interest in the scientific community due to its morphological and functional complexity, and from a phylogenetic point of view. A comparative approach has contributed to a better understanding of its physiology. Whereas the chemical nature of its secretions (mucous, serous or lipids) varies between different groups of tetrapods, the lipids represent the more common component among different species. Indeed, besides being an accessory to lubricate the nictitating membrane, the lipids may have a pheromonal function. Porphyrins and melatonin secretion is a feature of the rodent HG. The porphyrins, being phototransducers, could modulate HG melatonin production. The melatonin synthesis suggests an involvement of the HG in the retinal-pineal axis. Finally, StAR protein and steroidogenic enzyme activities in the rat HG suggests that the gland contributes to steroid hormone synthesis. Over the past twenty years, much has become known on the hamster (Mesocricetus auratus) HG, unique among rodents in displaying a remarkable sexual dimorphism concerning the contents of porphyrins and melatonin. Mainly for this reason, the hamster HG has been used as a model to compare, under normal conditions, the physiological oxidative stress between females (strong) and males (moderate). Androgens are responsible for the sexual dimorphism in hamster and they are known to control the HG secretory activity in different species. Furthermore, HG is a target of pituitary, pineal and thyroid hormones. This review offers a comparative panorama of the endocrine activity of the HG as well as the hormonal control of its secretory activity, with a particular emphasis on the sex dimorphic aspects of the hamster HG.

DAAM1 and PREP are involved in human spermatogenesis.

During differentiation of the male gamete, there is a massive remodelling in the shape and architecture of all the cells in the seminiferous epithelium. The cytoskeleton, as well as many associated proteins, plays a pivotal role in this process. To better characterise the factors involved, we analysed two proteins: the formin, dishevelled-associated activator of morphogenesis 1 (DAAM1), which participates in the regulation of actin polymerisation, and the protease, prolyl endopeptidase (PREP), engaged in microtubule-associated processes. In our previous studies we demonstrated their involvement in cytoskeletal dynamics necessary for correct postnatal development of the rat testis. Here, we used samples of testicular tissue obtained from infertile men by testicular sperm extraction and the spermatozoa of asthenoteratozoospermic patients. By western blot and immunofluorescent analysis, we found that DAAM1 and PREP expression and localisation were impaired in both the testis and spermatozoa, and in particular in the midpiece as well as in the principal and end-pieces of the flagella, as compared with spermatozoa of normospermic men. Our results provide new knowledge of the dynamics of spermatogenesis, raising the possibility of using DAAM1 and PREP as new markers of normal fertility.

Combined Raman and polarization sensitive holographic imaging for a multimodal label-free assessment of human sperm function.

Raman microspectroscopy (RM) and polarization sensitive digital holographic imaging (PSDHI) are valuable analytical tools in biological and medical research, allowing the detection of both biochemical and morphological variations of the sample without labels or long sample preparation. Here, using this multi-modal approach we analyze in vitro human sperm capacitation and the acrosome reaction induced by heparin. The multimodal microscopy provides morphofunctional information that can assess the sperms ability to respond to capacitation stimuli (sperm function). More precisely, the birefringence analysis in sperm cells can be used as an indicator of its structural normality. Indeed, digital holography applied for polarization imaging allows for revelation of the polarization state of the sample, showing a total birefringence of the sperm head in non-reacted spermatozoa, and a birefringence localized in the post-acrosomal region in reacted spermatozoa. Additionally, RM allows the detection and spectroscopic characterization of protein/lipid delocalization in the plasma and acrosomal membranes that can be used as valuable Raman biomarkers of sperm function. Interestingly, these spectral variations can be correlated with different time phases of the cell capacitation response. Although further experimentation is required, the proposed multimodal approach could represent a potential label-free diagnostic tool for use in reproductive medicine and the diagnosis of infertility.

Maternal non-Mendelian inheritance of a reduced lifespan? A hypothesis.

PURPOSE: A negative correlation exists between advanced maternal age and reproduction. Current data suggest that this correlation is due to a decline in oocyte quality with respect to female age. Since a new individual is derived from the fusion of a single sperm and egg, we tested whether the quality of this material could influence the long-term physiological health of offspring, by examining whether a link between parental age and lifespan of offspring exists. METHODS: We requested a search from the Swedish demographic database POPUM 3 maintained by the University of Umeå, Sweden between years 1700 and 1900. Parameters requested included mothers' and fathers' age at gestation, the lifespan of the children, cause of death of children and the region of birth. RESULTS: Complete data was obtained for 30,512 children born to 12,725 mothers and fathers. Kaplan-Meier estimators demonstrated a strong relationship between mother's age at gestation and the longevity of offspring. Extrinsic factors such as century of birth also had an effect on the data. The forward stepwise procedure on Cox's model of proportional hazards suggested that most significant intrinsic factors were mother's lifespan and mother's age at gestation. CONCLUSIONS: These data demonstrate that intrinsic and extrinsic factors influence the lifespan of children. Among intrinsic factors, mother's lifespan and age at gestation had a significant influence on the data. The influence of intrinsic factors remained significant despite a strong extrinsic influence. We suggest that the influence of the mother on the lifespan of offspring is due to extra-genomic factors.

First evidence of prothymosin α localization in the acrosome of mammalian male gametes.

Prothymosin α (PTMA) is a highly acidic intrinsically unstructured protein. Its expression in male gonads is evolutionary conserved; in rat testis it is specifically localized in the cytoplasm of post-meiotic germ cells, in association with the developing acrosome system. In the present paper we investigated on PTMA localization inside the head of mammalian spermatozoa (SPZ). We chose a confocal approach to ascertain whether PTMA is expressed in the acrosome or in the perinuclear theca, two regions that are tightly linked and partially overlapped in the mature haploid cells. The obtained results showed that PTMA is specifically localized in the acrosome of rat epididymal SPZ; the same experimental approach evidenced, for the first time, PTMA presence in human ejaculated SPZ. A Western blot analysis on protein extracts from human sperm head fractions confirmed the confocal data and demonstrated that the peptide is specifically associated with the inner acrosomal membrane fraction. Finally, when the acrosome reaction was induced in vitro by progesterone treatment on both rat and human sperm, PTMA signal was retained in the apical region of reacted SPZ. In conclusion, this study confirms the conservation of PTMA distribution in vertebrate male gametes and strongly supports a role for this polypeptide in their physiology.

Intracytoplasmic injection of morphologically selected spermatozoa (IMSI) improves outcome after assisted reproduction by deselecting physiologically poor quality spermatozoa.

PURPOSE: We used computer assisted sperm selection (MSOME) during cycles of intracytoplasmic sperm injection to test whether this technique improves results over traditional ICSI protocols. We also used the TUNEL assay to test whether MSOME could deselect physiologically abnormal spermatozoa. METHODS: Individual spermatozoa were examined with MSOME. Normal and abnormal spermatozoa were tested for the level of DNA fragmentation using TUNEL assay. In a prospective, randomized trial, patients were selected for standard ICSI, or IMSI techniques. We tested the two groups for biological and clinical parameters. RESULTS: 64.8% of spermatozoa, otherwise selectable for ICSI, were characterized by abnormalities after computer-assisted sperm analysis. These sperm were also characterized by an increase in the level of DNA fragmentation. We noted an increase in embryo quality, pregnancy and implantation rates after computerized sperm selection during ICSI procedures. CONCLUSIONS: Computerised selection of spermatozoa during ICSI procedures deselects physiological abnormal spermatozoa and improves clinical results.

Human cleavage-stage embryo vitrification is comparable to slow-rate cryopreservation in cycles of assisted reproduction.

OBJECTIVES: To compare embryo survival, pregnancy and implantation rates after cryopreservation of human cleavage-stage embryos with slow-rate cryopreservation or vitrification. STUDY DESIGN: 262 patients, attending for assisted reproduction, were prepared for oocyte retrieval using standard controlled ovarian hyperstimulation protocols. Excess embryos were cryopreserved on day 3 either by vitrification, or slow-rate cryopreservation in a programmable freezer. Cycles of thawing were monitored for thaw efficiency, pregnancy and implantation rates. RESULTS: Clinical pregnancy and implantation rates were highly comparable between cycles in which day 3 embryos were thawed either after slow-rate cryopreservation or vitrification. CONCLUSIONS: These data suggest that vitrification of human embryos during assisted reproduction cycles achieves comparable success rates to fresh cycles and therefore can be applied in the laboratory of assisted reproduction.

Mitochondria and human preimplantation embryo development.

Human reproduction, like all biological systems, is characterised by a large level of variability. In this field, the variability is observed as a large difference in implantation potential of human embryos developing in vitro, despite similarities in observable parameters such as rate of development and morphology of these embryos. One of the underlying factors that determines developmental potential in these embryos is the availability of energy in the form of ATP for development. Here, we suggest that, despite the evidence suggesting that mitochondrial metabolism is relatively inactive during preimplantation embryo development, aerobic (mitochondrial) metabolism contributes a major role in the supply of ATP. A second pathway, anaerobic respiration, is also active and the two pathways work in synchrony to supply all the ATP necessary. We discuss the differences in the two forms of energy production and suggest that, although anaerobic respiration can supplement deficiencies in the energy supply in the short term, this is not sufficient to substitute for aerobic respiration over long periods. Therefore, we suggest that deficiencies in the levels of aerobic respiration can explain variability in the implantation potential of apparently equivalent embryos.

An oocyte score for use in assisted reproduction.

PURPOSE: In this work, we describe a system for the morphological scoring of human oocytes prior to fertilisation and use this system to test whether oocyte morphology is an indicator of fertilisation, embryo development and implantation potential. METHODS: The study is a prospective trial of the use of oocyte morphological scores in 822 patients undergoing their first cycle of ICSI. Analyses of oocytes were performed prior to ICSI procedures and the scores compared with fertilisation rates, embryo quality and clinical results. RESULTS: 'Top quality' oocytes had a significantly higher level of fertilisation (96%) as compared to low scoring oocytes (25.6%). Where top quality oocytes formed top quality embryos, we noted a clinical success rate of 63.4%. CONCLUSIONS: Clinical success rates were increased in cases where top quality oocytes formed top quality embryos after ICSI. The analysis of oocyte morphology may represent a positive selection feature during ICSI.

The effect of extended culture of cumulus-oocyte complexes in follicular fluid during in vitro fertilisation cycles.

PURPOSE: To assess the clinical and biological effect of the preincubation of oocytes in follicular fluid prior to IVF and ICSI cycles. METHODS: A series of patients were treated by the preincubation of oocytes in the patients' follicular fluid for 3 h after oocyte retrieval followed by processing with standard protocols. Control oocytes were preincubated in normal IVF culture medium. Fluorescence techniques were used to examine oocyte mitochondrial membrane potential. RESULTS: Fertilisation, pregnancy, and implantation rates were all significantly improved after the preincubation of oocytes in follicular fluid. Further tests suggested that differences in pH between follicular fluid and artificial culture medium may be critical to these differences. CONCLUSIONS: Preincubation of human oocytes in follicular fluid improves the results after IVF. This may be partly due to the use of a non-"physiological" pH in artificial culture media during in vitro fertilisation procedures.

The maternal age effect: a hypothesis based on oxidative phosphorylation.

The 'maternal age effect' in human reproduction, characterized by a negative relationship between maternal age and reproductive efficiency, remains a poorly understood phenomenon. Current data suggest that oocyte physiology determines this relationship. In this review, we present a hypothesis of a mitochondrial role in the physiology of ageing in human oocytes. We suggest that the efficiency of oxidative phosphorylation in the ageing human oocyte is degraded by free radical attack on the primordial oocytes residing in the ovary. Although deficiencies in oxidative phosphorylation can be accounted for in the short term by anaerobic respiration, we suggest that, in the long term, the level of oxidative phosphorylation strongly influences oocyte quality.

Secondary male factor infertility after Roux-en-Y gastric bypass for morbid obesity: case report.

Surgical treatments such as the Roux-en-Y gastric bypass operation result in the successful treatment of morbid obesity; however, this type of operation may cause long-term side effects due to the reduced absorption of nutrients. Here, we present data suggesting that this operation can result in secondary infertility in males. Six healthy, previously fertile male subjects presented in our centre for secondary infertility after a Roux-en-Y gastric bypass operation for morbid obesity. Reproductive function was assessed with a series of spermiograms, and by testicular biopsy. Secondary azoospermy with complete spermatogenic arrest was diagnosed. The results suggest that weight reduction surgery may cause reproductive dysfunction.

Preimplantation genetic diagnosis for the treatment of failed in vitro fertilization-embryo transfer and habitual abortion.

OBJECTIVE: To apply preimplantation genetic diagnosis (PGD) for the treatment of patients with a history of failed IVF-ET or habitual aborters. DESIGN: Prospective clinical study. SETTING: Tertiary center for assisted reproduction. PATIENT(S): Ninety-four couples with failed IVF-ET after >2 IVF cycles and 64 couples with >2 spontaneous abortions. INTERVENTION(S): Patients were prepared for oocyte retrieval using standard controlled ovarian hyperstimulation protocols after standard laboratory techniques. Blastomeres from 6- to 8-cell embryos were analysed using fluorescence in situ hybridization with commercial chromosomal probes, and normoploid embryos were transferred on day 3 after fertilization. MAIN OUTCOME MEASURE(S): Pregnancy and implantation rates and live births. RESULT(S): Both 3- and 5-probe PGD resulted in a significantly higher outcome than controls for failed IVF-ET. Five-probe PGD appeared to be more suitable for habitual aborters. CONCLUSION(S): This pilot study suggests that 3-probe PGD is a valid option for failed IVF-ET patients. The use of five or more probes is indicated for habitual aborters.

Zygote versus embryo transfer: a prospective randomized multicenter trial.

PURPOSE: To determine the efficiency of transferring human zygotes as opposed to human day 2 or 3 embryos. METHODS: A prospective, randomized, Multicenter trial. Patients were randomized into zygote or embryo transfer. Patients were prepared for oocyte retrieval using standardized protocols. Oocyte retrieval was performed under general anesthesia. Oocytes and spermatozoa were treated using standard laboratory techniques. All protocols were coordinated by the coordinating center. RESULTS: A total of 386 patients were included in the trial. Pregnancy rates were 36.5% after zygote transfer and 42% after embryo transfer. Implantation rates were equivalent (17%) in both groups. CONCLUSIONS: No general difference was observed for zygote or embryo transfer. The results suggest that zygote transfer is a valid alternative to embryo transfer.

Histology, histochemistry, and ultrastructure of the harderian gland of the snake Coluber viridiflavus.

Analyses of the histology, histochemistry, and ultrastructre of the Harderian gland of Coluber viridiflavus prove the gland to be compound acinar and to produce a seromucous secretion. Acinar cells (type I) contain secretory granules that are composite, consisting ultrastructurally of three distinct parts that are sharply separated. They are similar to the "special secretory granules" described in the cells of the Harderian gland of the lizard Podarcis s. sicula. Some acini of the most anterior and posterior parts of the gland are mucous. Acinar cells (type II) of this type contain secretory granules that are Alcian blue/PAS positve. At the ultrastructural level, they appear homogeneous and of low density, characteristic of mucous secretions. These mucus-secreting anterior and posterior parts of the Harderian gland may by considered as regions of intial differentiation of the anterior and posterior lacrimal galnds.