An essential role for insulin and IGF1 receptors in regulating sertoli cell proliferation, testis size, and FSH action in mice.

Testis size and sperm production are directly correlated to the total number of adult Sertoli cells (SCs). Although the establishment of an adequate number of SCs is crucial for future male fertility, the identification and characterization of the factors regulating SC survival, proliferation, and maturation remain incomplete. To investigate whether the IGF system is required for germ cell (GC) and SC development and function, we inactivated the insulin receptor (Insr), the IGF1 receptor (Igf1r), or both receptors specifically in the GC lineage or in SCs. Whereas ablation of insulin/IGF signaling appears dispensable for GCs and spermatogenesis, adult testes of mice lacking both Insr and Igf1r in SCs (SC-Insr;Igf1r) displayed a 75% reduction in testis size and daily sperm production as a result of a reduced proliferation rate of immature SCs during the late fetal and early neonatal testicular period. In addition, in vivo analyses revealed that FSH requires the insulin/IGF signaling pathway to mediate its proliferative effects on immature SCs. Collectively, these results emphasize the essential role played by growth factors of the insulin family in regulating the final number of SCs, testis size, and daily sperm output. They also indicate that the insulin/IGF signaling pathway is required for FSH-mediated SC proliferation.

Clinical outcome after IMSI procedure in an unselected infertile population: a pilot study.

BACKGROUND: To date the IMSI procedure represents the only real-time and unstained method available to discard spermatozoa with ultrastructural defects. Several studies demonstrated that IMSI provides positive results in couples with severe male factor infertility or repeated ICSI failures. Aim of this pilot study is to evaluate the differences between IMSI and ICSI in terms of IVF outcomes in an unselected infertile patient population. METHODS: Three hundred and thirty-two couples were analyzed: 281 couples underwent conventional ICSI procedure and 51 underwent IMSI technique. RESULTS: No statistically significant differences were found between implantation rate (ICSI: 16,83%; IMSI: 16,67%), fertilization rate (ICSI: 77,27%; IMSI: 80,00%) and pregnancy rate (ICSI: 25,30%; IMSI: 23,50%). Both groups were comparable when considering live birth rate (ICSI: 11,39%; IMSI:13,72%), ongoing pregnancy rate (ICSI: 7,47%; IMSI: 5,88%) and miscarriage rate (ICSI: 17,78; IMSI: 5,26%). The subgroup analyses did not show a statistical difference between ICSI and IMSI neither in male factor infertility subgroup nor in patients with more than one previous ICSI attempt. A trend towards better laboratory and clinical outcomes was detected in the male factor infertility subgroup when IMSI was applied. CONCLUSIONS: Our preliminary results show that the IMSI technique does not significantly improve IVF outcomes in an unselected infertile population.

Morphological scoring of human pronuclear zygotes for prediction of pregnancy outcome.

BACKGROUND: As embryo selection is not allowed by law in Switzerland, we need a single early scoring system to identify zygotes with high implantation potential and to select zygotes for fresh transfer or cryopreservation. The underlying aim is to maximize the cumulated pregnancy rate while limiting the number of multiple pregnancies. METHODS: In all, 613 fresh and 617 frozen-thawed zygotes were scored for proximity, orientation and centring of the pronuclei, cytoplasmic halo, and number and polarization of the nucleolar precursor bodies. From these individual scores, a cumulated pronuclear score (CPNS) was calculated. Correlation between CPNS and implantation was examined and compared between fresh and frozen-thawed zygotes. The effect of freezing on CPNS was also investigated. RESULTS: CPNS was positively associated with embryo implantation in both fresh and frozen zygotes. With similar CPNS, frozen zygotes presented implantation rates as high as those of fresh zygotes. Nucleolar precursor bodies pattern and cytoplasmic halo appeared as the most important factors predictive of implantation for both types of zygotes, while pronuclei position was specifically relevant for frozen-thawed zygotes. Freezing induced an alteration of most zygote parameters, resulting in a significantly lower CPNS and a lower pregnancy rate. CONCLUSIONS: CPNS may be used as a single prognostic tool for implantation of both fresh and frozen-thawed zygotes. Lower CPNS values of frozen-thawed zygotes may also be indicative of freezing damage to zygotes. Successful implantation of frozen zygotes despite lower CPNS suggests that they may recover after thawing and in vitro culture.

Involvement of the pentose phosphate pathway and redox regulation in fertilization in the mouse.

Glucose metabolism is necessary for successful fertilization in the mouse. Both spermatozoa and oocytes metabolize glucose through the pentose phosphate pathway (PPP), and NADPH appears required for gamete fusion. The aims of this study were to further characterize the utilization of glucose by the fertilizing spermatozoon and the fertilized oocyte, to demonstrate the importance of the PPP in different steps of fertilization, and to examine whether the beneficial effect of glucose could be mediated by a NADPH-dependent enzyme involved in redox regulation. By using a fluorescent analog of 2-deoxyglucose, glucose uptake was evidenced in both the head and flagellum of motile spermatozoa. After sperm-oocyte fusion, an increase in glucose uptake by the fertilized oocyte was observed but not before the formation of the male and female pronuclei. By using a microphotometric technique, activity of glucose 6-phosphate dehydrogenase (G6PDH), the key enzyme of the PPP, was localized to the sperm head and midpiece. When epididymal spermatozoa were released into a glucose-containing medium, the NADPH/NADP ratio increased with capacitation. Sperm-oocyte fusion and meiosis reinitiation of the fertilized oocyte was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN); inhibition of sperm-oocyte fusion was relieved by NADPH. Sperm-oocyte fusion and meiosis reinitiation were also inhibited by diphenylamine iodonium, which is a flavoenzyme inhibitor reported to prevent reactive oxygen species (ROS) generation in mouse spermatozoa and embryos. These findings indicate that the PPP is involved in different steps of fertilization. Subsequent regulation of a NADPH-dependent flavoenzyme responsible of ROS production is envisaged.

What is the most relevant standard of success in assisted reproduction?: The cumulated singleton/twin delivery rates per oocyte pick-up: the CUSIDERA and CUTWIDERA.

National and international registries are essential tools for establishing new standards and comparing success rates, but they do not take into account the total pregnancy/delivery rate per oocyte recovery. In Switzerland and Germany, because of legal constraints, a maximum of three two-pronuclear zygotes are allocated for transfer whereas all the supernumerary pronuclear zygotes are immediately cryopreserved, preventing selection of the transferred embryos. We report on a 10 years' experience (1993-2002) of our centre which performs transfers of unselected embryos and cryopreservation at the two-pronuclear zygote stage. As approximately 30% of all deliveries are from cryo cycles, it is essential to take into account the contribution of the cryo transfers, and we propose therefore to evaluate, as a measure of IVF performance, the cumulated delivery rate per oocyte pick-up. This delivery rate is broken down further into the cumulated singleton delivery rate (CUSIDERA) and the cumulated twin delivery rate (CUTWIDERA). The sum (S) of these two rates is a measure of efficacy while the ratio CUTWIDERA/S as a percentage is a measure of safety of IVF treatments. Using these new indexes, the average 10 year efficacy and safety of our IVF programme were 26 and 19%, respectively. Both CUSIDERA and CUTWIDERA can be calculated easily in any clinical situation and yield useful parameters for patient counselling and internal/external benchmarking purposes.

Number of transferred embryos: how to reduce multiple pregnancies.

Because the diagnostic tools for predicting whether an early cleavage stage embryo can lead to a viable pregnancy are still elusive, transfer of more than one embryo remains quite common. However, the only way to reduce multiple pregnancies, considered as the main adverse effect of assisted reproductive technology, is to transfer a single embryo. In countries such as Switzerland and Germany, the law allows cryopreservation only at the 2-pronuclear stage. This restricts considerably the possibility of selecting the embryos to be transferred. Therefore, a good cryopreservation program at the 2-pronuclear stage is an essential tool to optimize the efficiency of in vitro fertilization (IVF). We therefore recommend the Cumulated Singleton Delivery Rate (CUSIDERA) as a measure of standard IVF efficiency. This rate averages approximately 23.5% when calculated over the last 10 years in our unit and reaches a value above 35% for patients with more than 10 zygotes. Elective single-embryo transfers and the decrease of iatrogenic multiple pregnancies in IVF remain dependent on better prognostic tools for the appropriate selection of patients, gametes, and zygotes.

Protein phosphorylation in mammalian spermatozoa.

Spermatozoa undergo a series of changes before and during egg binding to acquire the ability to fuse with the oocyte. These priming events are regulated by the activation of compartmentalized intracellular signalling pathways, which control the phosphorylation status of sperm proteins. Increased protein tyrosine phosphorylation is associated with capacitation, hyperactivated motility, zona pellucida binding, acrosome reaction and sperm-oocyte binding and fusion. The main tyrosine phosphorylated proteins during the course of capacitation and fertilization are localized to the flagellum, although tyrosine phosphorylation of less abundant proteins may also be regulated in the sperm head. Spermatozoa bound to the zona pellucida and fusing with the oocyte plasma membrane are characterized by a tyrosine phosphorylated flagellum. Protein phosphorylation in the flagellum is linked to hyperactivated motility in spermatozoa, but may also regulate additional functions involved in sperm-oocyte fusion. Factors involved in the appearance of phosphorylation more likely arise from the milieu surrounding the spermatozoa, but their uptake and processing are likely to be regulated differentially at specific steps within the female genital tract and during penetration of the egg vestments. One of these factors is glucose, the metabolic products of which (ATP and NADPH) appear to participate in signalling pathways by supporting a precise onset of tyrosine phosphorylation in the sperm flagellum leading to successful fertilization.