Intracytoplasmic spermatid injection and in vitro maturation: fact or fiction?

Intracytoplasmic injection with testicular spermatozoa has become a routine treatment in fertility clinics. Spermatozoa can be recovered in half of patients with nonobstructive azoospermia. The use of immature germ cells for intracytoplasmic injection has been proposed for cases in which no spermatozoa can be retrieved. However, there are low pregnancy rates following intracytoplasmic injection using round spermatids from men with no elongated spermatids or spermatozoa in their testes. The in vitro culture of immature germ cells to more mature stages has been proposed as a means to improve this poor outcome. Several years after the introduction of intracytoplasmic injection with elongating and round spermatids, uncertainty remains as to whether this approach can be considered a safe treatment option. This review outlines the clinical and scientific data regarding intracytoplasmic injection using immature germ cells and in vitro matured germ cells.

Ectopic porcine spermatogenesis in murine subcutis: tissue grafting versus cell-injection methods.

Fragments of testis tissue from immature animals grow and develop spermatogenesis when grafted onto subcutaneous areas of immunodeficient mice. The same results are obtained when dissociated cells from immature testes of rodents are injected into the subcutis of nude mice. Those cells reconstitute seminiferous tubules and facilitate spermatogenesis. We compared these two methods, tissue grafting and cell-injection methods, in terms of the efficiency of spermatogenesis in the backs of three strains of immunodeficient mice, using neonatal porcine testicular tissues and cells as donor material. Nude, severe combined immunodeficient (SCID) and NOD/Shi-SCID, IL-2Rgammacnull (NOG) mice were used as recipients. At 10 months after surgery, the transplants were examined histologically. Both grafting and cell-injection methods resulted in porcine spermatogenesis on the backs of recipient mice; the percentage of spermatids present in the transplants was 67% and 22%, respectively. Using the grafting method, all three strains of mice supported the same extent of spermatogenesis. As for the cell-injection method, although SCID mice were the best host for supporting reconstitution and spermatogenesis, any difference from the other strains was not significant. As NOG mice did not show any better results, the severity of immunodeficiency seemed to be irrelevant for supporting xeno-ectopic spermatogenesis. Our results confirmed that tubular reconstitution is applicable to porcine testicular cells. This method as well as the grafting method would be useful for studying spermatogenesis in different kinds of animals.

Generation of progeny from embryonic stem cells by microinsemination of male germ cells from chimeric mice.

Mice chimeric for embryonic stem (ES) cells have not always successfully produced ES-derived offspring. Here we show that the male gametes from ES cells could be selected in male chimeric mice testes by labeling donor ES cells or host blastocytes with GFP. Male GFP-expressing ES-derived germ cells occurred as colonies in the chimeric testes, where the seminiferous tubules were separated into green and non-green regions. When mature spermatozoa from green tubules were used for microinsemination, GFP-expressing offspring were efficiently obtained. Using a reverse study, we also obtained ES-derived progeny from GFP-negative ES cells in GFP-labeled host chimeras. Furthermore, we showed this approach could be accelerated by using round spermatids from the testes of 20-day-old chimeric mice. Thus, this technique allowed us to generate the ES cell-derived progeny even from the low contributed chimeric mice, which cannot produce ES-origin offspring by natural mating.

Intracytoplasmic spermatid injection can result in the delivery of normal offspring.

Almost one-third of all patients with nonobstructive azoospermia undergoing testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) have cancelled cycles due to failure to find spermatozoa. For these patients, every attempt should be made to rescue the cycles by searching for spermatids. In this retrospective study, we report our experience in using elongating (stage Sb2) and elongated (stage Sc and Sd1) spermatids for ICSI. The study included 488 consecutive ICSI and TESE cycles performed for 452 patients with nonobstructive azoospermia. In 179 (36.7%) cycles, neither spermatozoa nor mature spermatids (stage Sd2) suitable for injection were found. After an extensive search only Sb2, Sc, and Sd1 spermatids were found in 22 of these 179 cycles (12.3%). These spermatids were used for injection of retrieved oocytes. The fertilization rate was 33.2%, and 19 patients (86.4%) reached the embryo transfer stage. In 6 cycles a chemical pregnancy occurred, and 3 clinical pregnancies were established, resulting in the delivery of 3 healthy boys with normal karyotypes. When normal living spermatozoa or mature spermatids (stage Sd2) cannot be found during TESE, late spermatids (stage Sb2, Sc, and Sd1) can be used successfully and result in the delivery of healthy offspring.

Xenogeneic transplantation of human spermatogonia.

In the last 3 years, several studies have shown that xenogeneic transplantation of rodent spermatogonia is feasible. The treatment of infertile patients with spermatogenic arrest using the injection of immature germ cells has yielded only poor results. We attempted to establish a complete spermatogenetic line in the testes of mutant aspermatogenic (W/Wv) and severe combined immunodeficient mice (SCID) transplanted with germ cells from azoospermic men. Spermatogenic cells were obtained from testicular biopsy specimens of men (average age of 34.3 +/- 9 years) undergoing infertility treatment because of obstructive and non-obstructive azoospermia. Testicular tissue was digested with collagenase to promote separation of individual spermatogenic cells. The germ cells were injected into mouse testicular seminiferous tubules using a microneedle (40 microm inner diameter) on a 10 ml syringe. To assess the penetration of the cell suspension into the tubules, trypan blue was used as an indicator. Mice were maintained for 50 to 150 days to allow time for germ cell colonisation and development prior to them being killed. Testes were then fixed for histological examination and approximately 100 cross-sectioned tubules were examined for human spermatogenic cells. A total of 26 testicular cell samples, 16 frozen and 10 fresh, were obtained from 24 men. The origin of the azoospermia was obstructive (OA) in 16 patients and non-obstructive (NOA) in 8 patients. The concentration of spermatogenic cells in the OA group was 6.6 x 10(6) cells/ml, and 1.3 x 10(6) cells/ml in the NOA group (p < 0.01). The different spermatogenic cell types were distributed equally in the OA samples, ranging from spermatogenia to fully developed spermatozoa, but in the NOA group the majority of cells were spermatogonia and spermatocytes. A total of 23 testes from 14 W/Wv mice and 24 testes from 12 SCID mice were injected successfully, as judged by the presence of spermatogenic cells in histological sections of testes removed immediately after the injection. However, sections from the remaining testes examined up to 150 days after injection showed tubules lined with Sertoli cells and xenogeneic germ cells were not found. The reason why the two strains of mouse used as recipients did not allow the implantation of human germ cells is probably due to interspecies specificity involving non-compatible cell adhesion molecules and/or immunological rejection.

[Medically assisted reproduction with immature spermatozoa. Clinical examination and surgical technics]. / La procréation médicalement assistée avec spermatozoïdes immatures. Examens cliniques et techniques chirurgicales.

The authors report their experience with the use of spermatids in TESE programs where mature spermatozoa could not be isolated from testicular biopsies. The details of the indications for spermatid insemination, the technicity of the procedure and the results are exposed.

Use of immature germ cells for the treatment of male infertility.

Both animal experimentation data and preliminary clinical experience converge to suggest that normal progeny can be obtained by fertilizing oocytes with spermatids, the youngest male germ cells to have a set of haploid chromosomes. Spermatids can be obtained from the ejaculate of many patients with non-obstructive azoospermia. The use of ejaculated spermatids in the treatment of non-obstructive azoospermia is thus to be considered as an alternative to that of testicular spermatozoa. Fertilization with ejaculated spermatids makes it possible to avoid the potential adverse consequences of extensive testicular biopsy and may thus become the treatment of first choice. The recourse to testicular spermatids represents a treatment of last chance if no spermatids can be recovered either from the ejaculate and no spermatozoa from the testis.