No abstract

The aim of this work was to evaluate the relationship between follicular size at the time of oocyte retrieval, and the subsequent oocyte competence to be fertilized and to develop in vitro. All the obtained oocytes were classified according to the corresponding volume of aspirated follicular fluid. Aspirated volume of follicular fluid <2 ml corresponded to a follicular diameter <16 mm and constituted the small size group. Volume of follicular fluid from 2 to 6 ml corresponded to a diameter from 16 to 23 mm and constituted the medium size group. The large size group contained follicles with diameter >23 mm and corresponded to an aspirated volume of follicular fluid of >6 ml. A progressive and significant increase in the rates of oocytes with a first polar body was observed from the small size group to the other groups and from the medium to the large size group: 75.3, 85.9 and 95.3% respectively. After classical in-vitro fertilization (IVF), significantly better rates of fertilization and development were obtained in the medium size group compared to the two other groups. Moreover, a positive relationship was observed between follicular diameter and rates of embryos scored as 'good' when oocytes were fertilized by intracytoplasmic sperm injection (ICSI). These results demonstrated that follicular size is positively related to the oocyte ability to be fertilized and to develop. Although oocytes from small follicles gave lower percentages of development probably due to partial oocyte incompetence, they allowed an increase in the total number of embryos scored as 'good'.

Sometimes spermatozoa from ejaculate, epididymis or testis show a total absence of motility. For some patients, however, very few spermatozoa with very poor motility can be found after several hours of incubation (initially immotile spermatozoa). Other samples show no motility at all even after extended culture (totally immotile spermatozoa). Intracytoplasmic sperm injection (ICSI) is the only method available to select and retrieve a single immotile or initially immotile spermatozoon and inject it into the oocyte. A total of 103 patients with asthenozoospermia underwent ICSI in this study. It was shown that initially immotile and totally immotile spermatozoa, whatever their origin, have the capacity to fertilize an oocyte after ICSI. No significant difference could be observed between the fertilizing capacity of testicular or epididymal spermatozoa. Totally immotile ejaculated spermatozoa, however, fertilized significantly fewer oocytes after ICSI when compared with initially immotile ejaculated spermatozoa. Embryos of lower quality tended to be produced when totally immotile spermatozoa of any origin were used, compared with embryos resulting from initially immotile spermatozoa. Ongoing pregnancies were conceived after ICSI with initially immotile spermatozoa from any origin and totally immotile spermatozoa retrieved from testis only. One biochemical pregnancy was the result of embryo transfer after ICSI with totally immotile ejaculated spermatozoa. No supernumerary embryos could be cryopreserved for patients with totally immotile spermatozoa from ejaculate or epididymis. For a Kartagener patient, subzonal insemination (SUZI) seemed to be a better approach for obtaining fertilization and pregnancy than ICSI because no fertilization occurred after ICSI on sibling oocytes. Hence a healthy pregnancy was obtained after SUZI.

Spermatid microinjection into oocytes has proven to be a successful assisted reproduction procedure in the animal model and in the human species, since in the latter a few full-term pregnancies were actually obtained. Patients entering our spermatid injection study included those with a total absence of spermatozoa in the testicular tissue notwithstanding previous positive biopsies (n = 29): an obstructive problem (n = 3), secretory azoospermia (n = 26), and those with total arrest at the spermatogenesis level in previous explorative biopsies (n = 15). In the latter group, absence of spermatids was recorded in four cases. Mature, elongated, elongating and round spermatids (ROS) were injected in respectively 3, 2, 3, and 32 attempts. A total of 260 metaphase II oocytes were injected with ROS, 36 oocytes with spermatids at other stages of maturity. The rates of oocytes showing two pronuclei (2PN) and two polar bodies reached 22% and 64% respectively after injection of round or elongated-mature spermatids. The fertilization rate after ROS injection was influenced by the percentage of spermatozoa observed in a previous biopsy. Patients with a positive preliminary biopsy had significantly more 2PN (33%) when compared to those with a severe spermatogenic dysfunction and in whom no spermatozoa were found (only 11%) (P < 0.05). Incubation of oocytes in calcium ionophore after ROS injection had a positive effect on the rate of 2PN formation (36 versus 16%). Ninety per cent of all the normally fertilized oocytes cleaved. The percentage of grade A and B embryos depended on the type of injected cells: 12% after ROS and 30% with the other types of haploid cells. A total of 39 transfers resulted in five pregnancies: three full term with healthy babies delivered (one after ROS injection, and two after injection of an elongating and a mature spermatid), one 4 months ongoing (after elongating spermatid injection) and one miscarriage at 4 weeks (after elongated cell injection). Compared to our conventional intracytoplasmic sperm injection-testicular sperm extraction (ICSI-TESE) programme, the implantation rate after ROS injection was very low (5.5 versus 10.5%).

In the human, male ageing results in reproductive hormonal and cellular changes that can influence semen quality (volume, motility, concentration and morphology) and ultimately result in a reduced fertilising capacity and a longer 'time to pregnancy' for ageing men as well as an increased risk for miscarriage. This prospective cohort study of 278 patients undergoing a first in vitro fertilisation or intracytoplasmic sperm injection treatment was undertaken to examine whether patient's age was reflected in sperm motility, concentration, morphology as well as in DNA fragmentation (DFI) and immature chromatin (unprocessed nuclear proteins and/or poorly condensed chromatin) as measured by the sperm chromatin structure assay. This study also investigated the possible influence of male age (after correcting for female age) on their fertilising capacity, on obtaining a pregnancy and a healthy baby at home. Logistic regression analysis did not reveal any male age-related influences on sperm parameters like concentration, motility or morphology. No significant male age-related increase in DFI or immature chromatin was demonstrable for these patients. Elevated male age, after correcting for female age, was not related to lower fertilisation rates or significant decreases in the chance for a healthy baby at home.

There is good evidence in literature that intrauterine insemination (IUI) is the best first line treatment and most cost-effective procedure for moderate male factor subfertility. It seems very difficult to identify individual semen parameters predicting the likelihood of pregnancy after IUI. This can be explained by a lack of standardization of semen analysis, but many other methodological variables may also influence IUI success rates such as the patient selection, type of ovarian stimulation and number of inseminations per cycle. A review of the literature confirmed that sperm morphology using strict criteria and the inseminating motile sperm count (IMC) after sperm preparation are the two most important sperm parameters to assess the real impact of semen quality on IUI outcome. A universal threshold level above which IUI can be performed with acceptable pregnancy rates has not been determined yet, although IUI success seems to be impaired with <5% normal spermatozoa and an IMC of <1 x 10(6). Until now, no method of sperm preparation has been shown to be superior with regard to pregnancy rate after IUI. Whether supplementation of culture media with substances such as antioxidants and platelet activating factor may improve the results remains the subject of further research.

With the successful use of Assisted Reproduction, in particular intracytoplasmic sperm injection (ICSI), to treat infertile couples we have become less discriminating with the quality of spermatozoa we use to treat our patients. Numerous studies have shown the presence of nuclear DNA strand breaks in human ejaculated spermatozoa. The reason why human spermatozoa, in particular from men with abnormal semen parameters, possess these abnormalities in their nuclear DNA is still not clear. Two processes that have been linked to the presence of nuclear DNA strand breaks in spermatozoa are anomalies in apoptosis during spermatogenesis or problems in the packaging of the chromatin during spermiogenesis. Understanding the mechanisms responsible for producing abnormal spermatozoa in the human will improve our knowledge about certain causes of male infertility. More importantly, the impact of such sperm, if selected to perform ICSI, needs to be better understood so that any detrimental paternal effects can be avoided.

Thirty-two infertile couples with obstructive and non-obstructive azoospermia were included in this study. Testicular sperm extraction (TESE) was performed in 16 obstructive azoospermic cases where microsurgical sperm aspiration (MESA) or percutaneous sperm aspiration (PESA) were impossible because of totally destroyed epididymis and 16 non-obstructive azoospermia cases with severe spermatogenetic defect where the testicles were the only source of sperm cells. A total of 288 oocytes was obtained from 32 females and 84% were injected. The fertilization rates (FR) with 2 pronuclei (PN) and cleavage rate were 50.8 and 68.2% respectively. A total of 15 pregnancies was achieved (53% per embryo transfer), nine from the obstructive and six from the non-obstructive group. Four pregnancies resulted in clinical abortion (26.6%). The ongoing pregnancy rate was 39.2% per embryo transfer (ET) and 34.3% per started cycle. A high implantation rate was also achieved (26.6% in non-obstructive and 30% in obstructive azoospermia group). Using testicular spermatozoa in combination with ICSI in both obstructive and non-obstructive azoospermic groups, high implantation and pregnancy rates can be achieved.