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A simplified culture system was developed for the in-vitro maturation of early preantral mouse ovarian follicles. The follicles were cultured singly in 20 microliters droplets under oil in medium supplemented with recombinant follicle stimulating hormone (r-FSH) at 37 degrees C and 5% CO2 in air. The follicles grew and became attached to the bottom of the dish, progressively lost their spherical structure by outgrowth of the granulosa cells through the basal membrane and developed follicles with antral-like cavities. The normal three-dimensional follicular structure was lost but all components, i.e. theca, granulosa and oocyte, remained functional, as was proven by the oestradiol, inhibin and progesterone secretion patterns. Follicle survival exceeded 80% and histological analysis proved the absence of atresia and cell death in granulosa cells up to day 16. Oocytes of 55 (+/-4) microns diameter on the day of isolation reached 74 (+/-3) microns by day 16 of culture. The optimal moment for inducing the final meiotic maturation with human chorionic gonadotrophin was investigated: the highest absolute numbers of metaphase II oocytes were obtained on days 12 and 14 (39 and 41%). The fertilizing potential of the in-vitro matured oocytes was comparable to in-vivo matured controls. A 50% hatched-blastocyst development rate was observed.

Recovery of testicular spermatozoa from azoospermic patients with testicular failure followed by intracytoplasmic sperm injection (ICSI) is a recent advance in the treatment of male infertility. This study aimed at investigating which parameter(s) may predict successful testicular sperm recovery. We reviewed 395 testicular sperm recovery procedures and analysed the most frequently available parameters for clinical decision-making in azoospermic patients: (i) presence of at least one single spermatozoon in at least one preliminary semen analysis; (ii) maximum testicular volume; (iii) serum follicle stimulating hormone (FSH); and (iv) presence of spermatozoa in the histology of a randomly-taken testicular biopsy. Sensitivity, specificity, positive and negative predictive value, positive and negative likelihood ratio and accuracy were calculated for the above index parameters in different clinically relevant subgroups using receiver operating characteristic (ROC) curves whenever possible. Spermatozoa were always successfully recovered in patients with normal testicular histological findings (n = 173) or hypospermatogenesis (n = 16) but not in some patients with tubular sclerosis (seven out of 18), Sertoli cell-only pattern (55 out of 112) or maturation arrest (39 out of 76). Histopathology was the best test for predicting successful sperm recovery in the whole population (sensitivity: 86%, specificity: 93%, accuracy: 0.87). In patients with secretory azoospermia, histopathology was again the most accurate parameter (accuracy: 0.74), especially in patients showing Sertoli cell-only pattern (accuracy: 0.83) but not in patients showing maturation arrest (accuracy: 0.55). In patients with serum FSH concentrations > 12 IU/l and maximum testicular volume < 15 ml, histopathology was not found to be accurate. Semen analysis, maximum testicular volume and serum FSH were not highly predictive in all subgroups studied. Our analysis shows that no strong predictors for successful testicular sperm recovery are available except for testicular histopathology.

High success rates have been reported for the use of intracytoplasmic sperm injection (ICSI) in alleviating essentially andrological infertility. However, neither the relationship between any of the sperm parameters and the result of ICSI nor the minimal sperm requirements for ICSI have been investigated so far. In this paper, our objective was therefore to study the relationship between three basic sperm parameters (total sperm count, sperm motility and morphology) and the outcome of ICSI by retrospective analyses of fertilization, embryo development and pregnancy rates in 966 micro-injection cycles, performed with ejaculated semen. The results showed that there was no important influence from either the type or the extent of sperm impairment on the outcome of ICSI. Even in the most extreme cases of male-factor infertility, where cryptozoospermia or total astheno- or total teratozoospermia was diagnosed in the initial semen sample, high fertilization and pregnancy rates were obtained by ICSI. Only one condition had a strongly negative influence on the result of ICSI: where an immotile (presumably dead) spermatozoon was injected into the oocyte. Thus the only ultimate criterion for successful ICSI is the presence of at least one living spermatozoon per oocyte in the pellet of the treated semen sample used for micro-injection.