Results suggest that meiosis and spermiogenesis can be resumed in vitro, with normal differentiated spermatids showing a low fertilization potential but regular rates of blastocyst formation. However, most of the embryos did not reach the morula stage and showed major sex chromosome abnormalities.

Histopathological subgroups were positively correlated with successful gamete retrieval. No major outcome differences were observed between testicular sperm and elongated spermatids, either fresh or frozen-thawed. However, injection of intact round-spermatids showed very low rates of fertilization and no pregnancies.

Previous studies relating hormone and cytokine concentrations in follicular fluid to oocyte fertilizability were flawed by the uncertainty about the actual oocyte maturity status at the time of recovery and by the possible contribution of the male factor to failures of conventional in-vitro fertilization. This is the first study in which oocyte maturity was assessed immediately after recovery and only mature oocytes were selected for treatment by intracytoplasmic sperm injection. Fertilization outcomes were related to follicular fluid concentrations of 17beta-oestradiol, progesterone, follicle stimulating hormone, luteinizing hormone (LH), growth hormone (GH), prolactin (PRL), interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF alpha). Those oocytes that subsequently showed normal fertilization were harvested from follicles with higher concentrations of progesterone, GH, PRL, IL-1 and TNF alpha as compared with those of oocytes that failed to fertilize. Among the normally fertilized oocytes, low GH concentrations were associated with the failure of cleavage and with poor morphology of cleaving embryos, whereas rapidly cleaving embryos developed from oocytes recovered from follicles with high concentrations of LH and IL-1. These data suggest important roles for GH, IL-1 and TNF alpha, and of residual LH after pituitary suppression, as positive regulators of the final phase of oocyte intrafollicular development.

In an attempt to determine whether co-culture could promote sperm maturation, three patients with non-obstructive azoospermia, two with maturation arrest at the level of primary spermatocytes and one patient with <1% tubules showing complete spermatogenesis, and one patient with total globozoospermia, gave consent to experimentally co-culture round spermatids retrieved from the testicle on Vero cell monolayers. In all azoospermic patients elongating spermatids could be obtained from round spermatids. In one case of maturation arrest, of 37 round spermatids co-cultured for up to 5 days, 30% developed flagella, 46% matured to elongating and 19% to elongated spermatids, with one mature spermatozoon also obtained (3%). In the same patient, primary cultures of three round spermatids with flagella enabled development of one further mature spermatozoon. In the case with total globozoospermia, of six round spermatids co-cultured for up to 5 days, one mature spermatozoon was obtained, with a flagellum and normal head morphology. These preliminary findings suggest that it may be possible to overcome the round spermatid block, and even the triggering of morphological abnormalities arising at the spermiogenic level, by in-vitro maturation under special environmental conditions.

The incidence of Y/autosome translocations is low. Whereas involvement of non-acrocentric chromosomes often leads to infertility, cases related with acrocentric chromosomes are usually familial with no or minimal effect on fertility. A de novo (Yp/13p) translocation was found in a 32-year-old male referred for severe oligozoospermia. Conventional cytogenetic procedures (GTG, CBG and NOR banding) and molecular cytogenetic techniques (Fluorescence In Situ Hybridization, FISH) were performed on high-resolution chromosomes obtained after peripheral blood lymphocyte culture as also on interphase nuclei of spermatogenic cells from semen samples. Screening of AZF microdeletions in the Yq11.2 region known to be involved with spermatogenesis defects was also performed. GTG banding showed a (Yp/13p) translocation in all scored metaphases. CBG and NOR staining of the derivative chromosome revealed the maintenance of Yq heterochromatin and of the 13p NOR region. FISH with centromeric Y and 13/21 probes, SRY specific probe and X/Y (p and q arms) sub-telomeric probes gave the expected number/location of fluorescent signals. Hybridisation with a pan-telomeric repeat (TTAGGG) probe showed an absence of the telomeric sequences at the fusion point of the rearranged chromosome. FISH analysis with probes to chromosomes X, Y, 13 and 18 showed an abnormal segregation of the translocated chromosome during meiosis I, which explains that only 13.6% of the secondary spermatocytes were normal. Most of these became arrested, as after meiosis II the large majority of the round spermatids were normal (70%), as were in consequence most of the sperm (85.1%). Multiplex-PCR confirmed the intactness of the SRY region and showed absence of AZF microdeletions. We report a novel de novo (Yp;13p) translocation characterised by loss of the 13p and Yp telomeres. Meiotic studies using FISH demonstrated meiosis I chromosome unpairing and mal segregation that justifies the severe oligozoospermia. Although most sperm have a normal chromosomal constitution, preimplantation genetic diagnosis should be considered an option for this patient.

Late spermatids resulting from in-vitro culture of round spermatids in conditioned medium, either in controls in cases with a spermiogenetic block, appeared able to successfully fertilize the human oocyte and elicit normal embryo development.

In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.

We present nine cases of spermatid intracytoplasmic injection for the treatment of non-obstructive azoospermia. In eight cases, no elongated spermatids or spermatozoa were found in previous spermiograms or testicular biopsies. In these patients, treatment was performed using ejaculated (n = 6) and testicular (n = 2) retrieved round spermatids (Sa type). In cases where ejaculated round spermatids were used, they were isolated on the day before oocyte retrieval and left in culture for 24 h before intracytoplasmic sperm injection (ICSI). No pregnancy was obtained in either group, although culturing seemed to increase the fertilization rate. In one other case, elongated spermatids were observed in the previous spermiogram and thus a normal ICSI procedure was scheduled. However, on the day of oocyte retrieval, no spermatids could be recovered from fresh sequential ejaculates, and a testicular open biopsy was then performed. Both round and elongated spermatids were found in the testicular tissue, but only the more mature germinal cells (Sb2) were injected. From this case, a normal pregnancy was obtained which resulted in the birth by Caesarean section at 37 weeks of gestation of a normal healthy baby girl, weighing 2700 g.