[Comparison of two methods of cryoconservation of sperm when in very small numbers]. / Comparaison de deux méthodes de cryoconservation de spermatozoïdes en très petit nombre.

OBJECTIVE: In case of severe oligozoospermia or following testicular biopsy in case of azoospermia, fertilization remains possible by intracytoplasmic sperm injection (ICSI). However, if there are only a few spermatozoa available to be preserved for a delayed ICSI, these gametes must be cryopreserved according to a specific method. We have found that, using the straw classical technique, less than 2 sperm were recovered at thawing when 20 sperm were frozen and no sperm were available following freezing-thawing of only 1 to 10 sperm per straw. Then, the feasibility of programmed ICSI is always uncertain in these cases and unnecessary medical acts (ovarian stimulation and egg collection) as well as patient disappointment are frequent. MATERIAL AND METHODS: We have tested a new technique by introducing (with an ICSI pipette) 1 to 100 sperm in a microdrop (0.5 microl) of freezing medium setting in a culture dish under paraffin oil. RESULTS: Following freezing-thawing the dish, all (100%) of the frozen spermatozoa were recovered. DISCUSSION AND CONCLUSION: Since only motile sperm were frozen, the sperm which are non motile after thawing (more than 50%) are nevertheless considered usable for ICSI.

Flow cytometry isolation and reverse transcriptase-polymerase chain reaction characterization of human round spermatids in infertile patients.

Flow cytometry coupled to cell sorting is proposed as a method to isolate round spermatids from testicular biopsies in obstructive azoospermic patients. The cells were separated on the basis of their size and density only. We obtained homogenous populations of alive round spermatids free of lymphocytes and diploid germ cells. The detection of protamine 1 gene (PRM1) and PRM2 expression in the sorted cells proves that these cells are round spermatids. On the contrary, neither the expression of CD3-delta, which is specific to lymphoid cells, nor that of MAGE1, which has been demonstrated in diploid germ cells, could be observed in the round spermatid population even after using a nested polymerase chain reaction (PCR) assay. The flow cytometry procedure failed to isolate round spermatids from ejaculates in non-obstructive azoospermic patients. In > 39 ejaculates tested by reverse transcriptase-PCR, only nine revealed the presence of some round spermatids, as demonstrated by the expression of PRM1. However, these round spermatids did not express PRM2.

Delayed embryo transfer in an in-vitro fertilization programme: how to avoid working on Sunday.

It has previously been demonstrated that use of gonadotrophin releasing hormone analogues allows the administration of HCG to be delayed and makes it possible to avoid oocyte retrievals at weekends. In this study, we demonstrate that it is also possible to avoid embryo transfers on Sundays, by delaying transfers of Friday retrievals until Monday, without any apparent change in the results. This facility should improve the cost-effectiveness of assisted reproduction programmes.

An improved use of buserelin in ovarian stimulation for in-vitro fertilization.

In earlier IVF programmes, subcutaneous buserelin (Suprefact, Hoechst) was initially administered three times per day (200 micrograms x 3); then twice daily (300 micrograms x 2). We now suggest that a single administration of 600 micrograms daily may be equally effective. In a preliminary study, 20 patients were selected on the basis of tubal or idiopathic infertility and received 0.6 ml buserelin subcutaneously once a day, beginning on day 1 or 2 of the cycle. A sufficient pituitary desensitization was obtained on day 10 in 75% of patients and on day 16 for 100% and the ongoing pregnancy rate was 35% per treatment cycle. A randomized study comparing the effect of 600 micrograms of buserelin administered in one (n = 50) or two injections (n = 46), has been carried out and indicates that the results in terms of the ovarian suppression and pregnancy rates, were similar. Therefore, this protocol represents a simplification of the treatment with buserelin.