Development to blastocyst is impaired when intracytoplasmic sperm injection is performed with abnormal sperm from infertile mice harboring a mutation in the protein phosphatase 1cgamma gene

Idiopathic azoospermia, characterized by abnormal spermatogenesis, is commonly treated by performing intracytoplasmic sperm injection (ICSI) with sperm retrieved from testicular biopsies. However, no controlled experiments have been performed using an animal model to assess the efficacy or safety of the procedure. We have performed ICSI with testicular sperm obtained in a similar manner from testes of male mice homozygous for a null mutation in the protein phosphatase 1cgamma gene (PP1cgamma) or those of their wild-type littermates. PP1cgamma mutant testicular sperm are less resistant to sonication than are wild-type sperm and display a range of morphological abnormalities, similar to those reported for testicular sperm from idiopathic azoospermic men. PP1cgamma mutant sperm are unable to support development to the blastocyst stage, resulting in arrested development either before or just after compaction. A comparison of testicular and epididymal sperm from wild-type males revealed that the epididymal sperm caused embryos to fragment at an elevated rate. These results suggest that ICSI with any kind of testicular sperm carries an increased risk of embryo fragmentation and that abnormal testicular sperm has an added risk of embryo wastage at later preimplantation stages.     

Nouveaux marqueurs séminaux

Several proteins detected in seminal plasma have been proposed as prognostic factors in patients with secretory azoospermia. These proteins can provide useful information about the state of spermatogenesis in cases of testicular origin. Anti-Müllerian Hormone (AMH) is strictly derived from Sertoli cells and, in adult males, is preferentially secreted into the seminiferous tubule. The seminal AMH concentration has been found to be correlated with spermatogenesis. It may be a useful prognostic factor in the case of secretory azoospermia before testicular sperm recovery for ICSI.     

The treatment of obstructive azoospermia by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) allows the treatment of virtually every type of male infertility. Unlike in vitro fertilization (IVF), its success does not depend on sperm concentration, motility or morphology and most of the physical barriers to fertilisation are by-passes. Since ICSI does not require strongly motile sperm, its use has now been expanded to incorporate immature sperm from the testes and epididymides. Successful fertilisation, pregnancies and healthy babies have all been reported. However, concerns about the safety of ICSI remain due to its short clinical history and the lack of testing on animal models. Male fertility potential for assisted reproduction by ICSI cannot be measured by conventional parameters. Sperm DNA integrity is increasingly recognised as a more useful indicator. Studies have shown that sperm with higher levels of DNA damage have lower fertilisation rates after IVF and ICSI. Sperm with DNA damage above a certain threshold are associated with a longer time to conceive in otherwise apparently fertile couples and a higher miscarriage rate. DNA damage has been shown to be associated with impaired embryo cleavage. Our group has shown that sperm DNA from testicular sperm is less fragmented than that from epididymal sperm and suggest its preferred use in ICSI. In addition to nuclear (n) DNA we also assessed the quality of mitochondrial (mt) DNA from testicular sperm from men with obstructive azoospermia undergoing ICSI. We observed that couples achieving a pregnancy had both less mtDNA deletions and less nDNA fragmentation. We found inverse relationships between pregnancy and sperm mtDNA deletion numbers, size and nDNA fragmentation. No relationships were observed with fertilisation rates. With this knowledge, we designed an algorithm for the prediction of pregnancy based on the quality of sperm nDNA and mtDNA. Each year 40,000 men have a vasectomy in the UK but every year 2500 request a reversal to begin a second family. For such men, vasectomy reversal has recently been replaced in part by testicular biopsy via fine-needle testicular sperm aspiration (TESA) or percutaneous epididymal sperm aspiration (PESA) performed at an outpatient clinic and subsequently used in ICSI. Since these were previously fertile men it has been assumed that they had ‘fertile’ sperm. However the assited conception success rates of these mens partners has not been assessed until recently. We have shown a significant reduction in the clinical pregnancy rates in the partners of men who had had a vasectomy ≥10yrs previously. There is also evidence to suggest that spermatogenesis is significantly impaired in vasectomised men. Marked decreases in spermatocytes, spermatids and spermatozoa have been observed. We have found this to be associated with concomitant increases in apoptotic markers, such as Fas, FasL and Bax. The quality of the remaining sperm is also compromised. Sperm DNA from vasectomized men shows substantial damage which increases with time after surgery. This new use of ICSI will be discussed.     

Germ-cell nondisjunction in testes biopsies of men with idiopathic infertility.

Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis.     

Qualité du gamète testiculaire

The combination of testicular sperm extraction (TESE) and ICSI has constituted a breakthrough in the treatment of men with nonobstructive azoospermia. However, impaired spermatogenesis raises the question of the quality of testicular spermatozoa obtained by testicular biopsy, when considering the male gamete as the product of a sequential and programmed spermatogenic process. The aim of this paper is: 1) To briefly recall the successive events of sperm protein synthesis; 2) To define the characteristics of sperm cells retrieved from the testis and 3) To evaluate the effect of nuclear anomalies on the outcome of ICSI.     

Syndrome de klinefelter et conseil génétique

Klinefelter’s syndrome is a common sex chromosomal aberration generally characterized by hypergonadotrophic hypogonadism and azoospermia. However, spermatogenesis impairment is variable and severe oligozoospermia can be found in some men, particularly those exhibiting a mosaic karyotype 47,XXY/ 46,XY. New reproductive technologies, such as intracytoplasmic sperm injection (ICSI), allow Klinefelter patients to have a progeny, even those who are azoospermic after testicular sperm recovery. The question therefore arises of whether or not there is a genetic risk for pregnancies from affected fathers. Sperm karyotyping, by in vitro penetration of zona-free hamster eggs or by fluorescence in-situ hybridization (FISH), is a method of choice for measuring aneuploidy rate in spermatozoa of patients carrying gonosomal abnormalities. A theoretical model would predict a high level of 24,XX and/or 24,XY disomic sperm cells in Klinefelter patients if 47,XXY spermatogonia were able to complete meiosis and achieve spermatogenesis. Interestingly, current observations show that the rate of abnormal spermatozoa in these patients is low, around 1–2%, which indicates that only 46,XY spermatogonia can produce mature sperm cells and that oligozoospermic Klinefelter patients probably carry a 47,XXY / 46,XY mosaicism, at least at the testicular level. However, this low but statistically significant level of disomic spermatozoa emphasizes the fact that their spermatogenesis occurs in a compromised environment which could increase the risk of meiotic errors. Therefore, the possible occurrence of autosomal aneuploidies in children born from Klinefelter fathers leads to the following recommendations: a) individual analysis by FISH of the sperm aneuploidy rate in each Klinefelter patient candidate for ICSI; b) proposal of fetal karyotyping after amniocentesis in pregnancies obtained by this technique.     

Devant une azoospermie ou une oligozoospermie extrême: ICSI ou IAD?

Intra-cytoplasmic sperm injection (ICSI) allows to obtain evolutive embryos even in case of extreme oligozoospermia: this technique must thereby be proposed to couples before considering artificial insemination with donor semen (AID). Azoospermia sets some specific problems, particularly in case of deep alteration of spermatogenesis except in rare seminiferous tubules. There is a high risk of ICSI failure because of absence of spermatozoa extracted from testicular biopsies. AID represents therefore a much less heavy solution. In every cases, the opinion of the couple is very important to take in account.     

Poor centrosomal function of cat testicular spermatozoa impairs embryo development in vitro after intracytoplasmic sperm injection

In the domestic cat, morula-blastocyst formation in vitro is compromised after intracytoplasmic sperm injection (ICSI) with testicular compared to ejaculated spermatozoa. The aim of this study was to determine the cellular basis of the lower developmental potential of testicular spermatozoa. Specifically, we examined the influence of sperm DNA fragmentation (evaluated by TUNEL assay) and centrosomal function (assessed by sperm aster formation after ICSI) on first-cleavage timing, developmental rate, and morula-blastocyst formation. Because the incidences of DNA fragmentation were not different between testicular and ejaculated sperm suspensions, DNA integrity was not the origin of the reduced developmental potential of testicular spermatozoa. After ICSI, proportions of fertilized and cleaved oocytes were similar and not influenced by sperm source. However, observations made at 5 h postactivation clearly demonstrated that 1) zygotes generally contained a large sperm aster after ICSI with ejaculated spermatozoa, a phenomenon never observed with testicular spermatozoa, and 2) proportions of zygotes with short or absent sperm asters were higher after ICSI with testicular spermatozoa than using ejaculated spermatozoa. The poor pattern of aster formation arose from the testicular sperm centrosome, which contributed to a delayed first cleavage, a slower developmental rate, and a reduced formation of morulae and blastocysts compared to ejaculated spermatozoa. When a testicular sperm centrosome was replaced by a centrosome from an ejaculated spermatozoon, kinetics of first cell cycle as well as embryo development quality significantly improved and were comparable to data from ejaculated spermatozoa. Results demonstrate for the first time in mammals that maturity of the cat sperm centrosome (likely via epididymal transit) contributes to an enhanced ability of the spermatozoon to produce embryos that develop normally to the morula and blastocyst stages.     

La fécondation par injection d'une spermatide dans l'ovule

There was a recent large spreading of intracytoplasmic sperm injection (ICSI) to treat male infertility in most of in vitro fertilization (IVF) laboratories. The recent data confirm the efficacy of ICSI even by using testicular sperm or sperm with grossly abnormal phenotype (round head, absence of motility). Moreover it appears that ICSI could pass beyond the last events of spermatogenesis (i.e. spermiogenesis), since normal development follows fertilization with the male gamete, spermatid, recovered just after completion of meiosis. It is obvious that the natural properties of a mature spermatozoon (motility, ADN compaction, oocyte recognition and penetration) are only necessary to reach the site of fertilization (into the female tube) and to pass through the protective enveloppes around the oocyte (cumulus oophorus, zona pellucida, plasma membrane). The current view that spermatids lack genetic maturation comparing to eggs is not valid since eggs are only secondary oocytes at a meiotic stage equivalent to that of secondary spermatocytes. Moreover genetic imprinting occurs before meiosis, and cytoplasmic structures which seem necessary for embryo development are already present in spermatids. ICSI using spermatid cells is relevant to men suffering non obstructive azzospermia if spermatids are recovered from either the ejaculate or the testicular tubes. Several normal babies were born after injection of round spermatids. Since these spermatogenic cells are present in the ejaculate of most of the patients with non obstructive azoospermia (76% in our lab), one can estimate to 5–10% the proportion of sterile men potentially concerned by conception with spermatids. However certain of these men may have occasional sperm found with testicular sperm extraction and it is to early to know if such iatrogenic extraction is always preferable to ejaculate spermatid collection.     

The development of regionalized lipid diffusibility in the germ cell plasma membrane during spermatogenesis in the mouse

The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed.     

Facteurs paternels influençant le développement embryonnaire

The influence of sperm factors on early human embryonic development has been shown through the production of lower quality embryos in case of in vitro fertilization (IVF) for male infertility when compared to tubal infertility. Numerous factors may be implied in this effect. Indeed, zygotic centrosome, cellular organizer having a key role in mitosis, is exclusively from paternal origin and some IVF failures have correlated to centrosome deficiencies. A sperm cytosolic protein, oscillin, induces oocyte activation after sperm-egg fusion and could be responsible of clivage retardations observed in sperm abnormalities. Paternal chromosomal abnormalities, shown in as much as 8% of severe oligospermia, could lead to development blockage of the embryos. Last, genome imprinting during spermatogenesis, could be impaired in testicular dysfunctions, and thus the embryos could have low developmental abilities. However, it must be pointed out that no influence of sperm quality has been found using intracytoplasmic sperm injection (ICSI) procedures. Thus we can postulate that this technique alleviate the problem found with IVF. Thus the factor involved in the paternal effect on early embryogenesis is likely oocyte activation     

Spermatogenesis parameters and testosterone production at puberty as predictors of testicular functional activity in mice (<Emphasis Type="Italic">Mus musculus</Emphasis>)

The aim of this study was to investigate fertility-associated parameters of spermatogenesis and androgenic status in male laboratory mice at puberty and to assess their prognostic significance in the realization of the definitive testicular function. In three inbred murine strains, BALB/cLac, CBA/Lac and PT, the serum testosterone level, its testicular concentration, epididymal sperm count (sperm reserve) and portion of sperm with abnormal head morphology were evaluated on days 45 (puberty) and 90 (adulthood) of postnatal development. CBA/Lac males were characterized by a lower epididymal sperm count vs. other strains at both ages indicative of poorer spermatogenesis. At the same time, CBA/Lac males had a lower portion of sperm with abnormal head morphology, and this could be considered as a compensatory reaction aimed at improving sperm fertility. Distinct inter-strain differences in the portion of sperm with morphologically abnormal heads were established at both ages, while the inter-strain ratio remained invariable (BALB/cLac > PT > CBA/Lac). Thus, the level of abnormal spermatogenesis in the pubertal period may have a predictive significance for the definitive testicular activity in adult mice. No inter-strain and age-dependent changes were found in serum and testicular testosterone levels except for the PT strain, in which both testosterone levels rose from puberty to adulthood, suggesting a shift of the pubertal testosterone peak towards later times. Our data show that in male laboratory mice the genetic peculiarities of the testicular function manifest themselves during puberty and persist until adulthood.     

Les spermatozoïdes immobiles frais ou décongelés en fécondation assistée

Contrasting with sperm count or morphology, complete lack of mobile sperm may seriously impair ICSI fertilization and pergnancy rate. In three cases with flagellar skeleton abnormalities [dynein arm absence] only immobile sperm were found in the ejaculate. Following repeated ejaculations, higher rates of viable spermatozoa and even some motile spermatozoa could be found. Some times, in nonobstructive azoospermia, extensive sperm search didn't allow us to find but immobile sperm mostly, with very few motile sperm cells, not enough for the microinjection of all oocytes. The third group of immobile sperm is iatrogenic, following freezing and thawing surgically retrieved, testicular or epididymal spermatozoa in order to avoid repeated surgical retrieval. Following thawing, one find frequently very few motile spermatozoa that may be not enough for all retrieved oocytes and it might be necessary to inject some eggs with immobile spermatozoa. The outcome of ICSI using mobile and immobile sperm was compared in the three above mentioned groups: 1-immobile ejaculated sperm with flagellar defects, 2-immobile sperm discovered in the ejaculate after extensive sperm search and 3- immobile frozen-thawed testicular or epididymal spermatozoa. The results of ICSI in these groups show that fertilizing ability of fresh or frozenthawed immobile spermatozoa is not significantly different from ICSI with mobile sperm from the same origin. More over, in the first group with flagellar abnormalities, repeated ejaculations allowed us significantly increase sperm viability and fertilization ability. Finding only immobile fresh or frozen-thawed sperm the day of egg retrieval should not lead us to ICSI cancellation. Pregnancies may occur with such immobile sperm.     

Fertilité et aneuploïdies spermatiques après traitement par radiothérapie et/ou chimiothérapie pour cancer du testicule ou lymphome

Improvements in cancer therapy have considerably modified patient survival rates over recent years. However, the side effects of these treatments especially the effects on fertility, must be taken into account. Anticancer therapy can transiently inhibit spermatogenesis. Factors such as pretreatment semen parameters and the type of chemotherapy or radiotherapy may influence recovery of spermatogenesis, but it is still impossible to predict the probability of and time to recovery for each patient. Sperm banking remains the only way to prevent the effects of cancer treatment on male fertility. Another possible effect of chemotherapy or radiotherapy is genetic damage to germ cells. For instance, chromosomal abnormalities in viable sperm produced by these patients after recovery of spermatogenesis may result in fetal death or congenital abnormalities in their offspring. It has been fairly well documented that, during the first three months after treatment, DNA breaks and abnormal chromosomal segregation induced by chemotherapy/radiotherapy lead to structural and numerical chromosomal abnormalities in spermatozoa, respectively. However, the long-term effects on genetic sperm content have not been clearly established. The results of published studies are contradictory and are based on limited numbers of patients (maximum of 6). We present the preliminary results of a retrospective study concerning patients treated for testicular cancer or lymphoma between 1995 and 2000. Fluorescence in situ hybridization (FISH) analysis of chromosomes X, Y and 18 was performed on sperm collected one to five years after treatment and compared to the data obtained for non-affected fertile men. For four out of 13 patients, we found a significantly increased frequency of aneuploidy rates (mainly XY disomy and diploidy), and these results did not appear to be correlated with sperm count, sperm morphology or post-treatment duration. In conclusion, increased sperm aneuploidy rates appear to only concern a small number of patients, to varying degrees and without any predictive factors. According to published data and our preliminary results, we recommend waiting at least two years before starting ART (Assisted Reproduction Therapy) for patients treated for testicular cancer or lymphoma. Moreover, FISH analysis could be helpful to choose between ART with post-treatment sperm or cryopreserved sperm.     

Experimental mild increase in testicular temperature has drastic,but reversible,effect on sperm aneuploidy in men: A pilot study

In mammals testicular and epididymal temperature increase impairs spermatogenesis. This experimental study investigates the effects of a mild testis temperature increase (i.e. testis temperature remains below core body temperature) on sperm aneuploidy in men. In 5 fertile volunteers a testicular temperature increase was induced by maintaining the testes at suprascrotal position using specially designed underwear for 15 ± 1 h daily for 120 consecutive days. After heating men were followed for next 180 days. A control group (27 men) was recruited. Semen samples were collected before, during and after heating period and analyzed for chromosomes X, Y and 18 for aneuploidy using FISH. A total of 234,038 spermatozoa were studied by FISH. At day 34 of heating, mean sperm aneuploidy values were not modified. From day 34 of heating until day 45 post heating, FISH evaluation was not possible due to the drastic fall of sperm count. At day 45 post-heating total sperm aneuploidy percentage was twice higher than before heating whereas. Sex disomy (sperm XY18), sex chromosome nullisomy (sperm 18) were significantly higher than controls. These effects were completely reversed at 180 days post heat exposure. Conclusion: A mild rise in testicular temperature significantly increases sperm aneuploidies, reflecting an effect on the meiosis stage of spermatogenesis. The effect of heating was reversible and suggests that recovery of aneuploidy to normal values requires at least two cycles of spermatogenesis. Nonetheless, the low number of volunteers was a limitation of this pilot study and warrants further research on larger population.     

Generation of normal progeny by intracytoplasmic sperm injection following grafting of testicular tissue from cloned mice that died postnatally

Animal cloning by nuclear transfer has been successful in several species and was expected to become an alternative reproductive technique. Among the problems associated with this cloning technique, however, are its low success rate and high mortality of cloned animals even if they develop to term. Nuclear transfer has thus come to be considered too difficult to apply as a reproductive technique. The transplantation of male germ cells or pieces of testicular tissue has enabled the induction of spermatogenesis from fetal or postnatal male mice. In the present study, we examined whether functional male gametes could be obtained by the transplantation of pieces of testicular tissue from cloned mice that died immediately after birth with typical aberrant phenotypes, such as large offspring syndrome. Donor testicular tissues were retrieved from cloned mice that died postnatally and were transplanted into the testes of recipient nude mice. Two to three months after transplantation, the grafted donor testicular tissue had grown in the host testis, and histological analysis showed that spermatogenesis occurred within the graft. Intracytoplasmic sperm injection demonstrated that the testicular sperm generated in the grafted donor tissue were able to support full-term development of progeny. These results clearly showed that functional spermatogenesis could be induced by transplanting testicular tissue from cloned mice that died postnatally into recipient mice. The strategy presented here will be applicable to cloned animals of other species, because the xenografting of testicular tissue into mice has been demonstrated previously to be possible.     

Les prélèvements testiculaires dans les azoospermies sécrétoires: le point de vue du Biologiste de la Reproduction sur la lecture du prélèvement

The prognosis of severe male sterility (nonobstructive azoospermia) has considerably improved over recent years with the introduction of ICSI. The human reproduction biologist actively collaborates with the surgeon in the search for and extraction of sperm from testicular tissue (TESE). The presence of the biologist during surgery is mandatory to guide exploration and to avoid an excessive number of biopsies. Sperm extraction is performed in the laboratory by mechanical extraction with fine forceps. Enzymatic extraction with collagenase IV can be used in cases of nonobstructive azoospermia to optimize tissue dispersion and to improve retrieval of mature cells. The use of only one fraction (50%) of Pure Sperm (JC Diffusion, Lyon France) limits the loss of spermatozoa. to optimize the results in cases of akinesia, it may be useful to induce sperm motility by pentoxifylline or perform in vitro culture for three days. Sperm cryopreservation is compulsory in the case of nonobstructive azoospermia. This allows programming of TESE for a different time from that of ICSI. All results obtained with cryopreserved testicular sperm, in recent years, are encouraging. It is also recommended to freeze several small fractions of sperm in order to limit the number of surgical procedures on the testicle. This article presents the results of our experience in 36 cases of nonobstructive azoospermia. Extraction was negative in 13 cases (36%), similar to the rate reported in the literature.     

Utilisation des spermatozoïdes testiculaires en ICSI. Intérêt de la culture in vitro. Revue de la littérature

The number of ICSI cycles performed with testicular spermatozoa has increased dramatically over recent years. However, one of the technical limitations of this approach concerns the extremely reduced motility of testicular spermatozoa. However, increased sperm motility was observed after incubating testicular samples for several hours. Therefore, in order to improve ICSI success rates, several authors have tested the effect of previous in vitro culture. We present a review of the literature on this subject. In vitro culture does not appear to be very useful in cases of obstructive azoospermia, as, apart from possible sperm “maturation” during this culture phase, a high proportion of motile spermatozoa is usually already observed prior to in vitro culture. The benefits of in vitro culture appear to be greater in the case of non-obstructive azoospermia, as when spermatozoa are present on the biopsy, they are usually immobile. However, discordant results have been published: after in vitro culture, spermatozoa have been reported to be either motile or mostly dead. Regardless of the type of azoospermia, the best results are obtained after 3–4 days of in vitro culture. Addition of recombinant FSH to the culture medium also appears to be effective. Cryopreservation of testicular biopsies may also be associated with in vitro culture and the in vitro culture/freezing sequence appears to give better results than the freezing/in vitro culture sequence. Very few studies have reported the results of ICSI using frozen in vitro cultured spermatozoa, as most published studies concern fresh spermatozoa, used after 1–2 days of in vitro culture with satisfactory fertilization and pregnancy rates. In vitro culture of testicular spermatozoa may therefore constitute an interesting research approach to improve the results of ICSI when the number of spermatozoa and/or motility are very low. In addition, in vitro culture of testicular spermatozoa appears to be a good tool to study the mechanisms of acquisition of motility, which are still poorly understood.     

Progeny from sperm obtained after ectopic grafting of neonatal mouse testes

Ectopic grafting of testicular tissue is a promising new approach that can be used to preserve testicular function. This technique has been used recently to differentiate the neonatal testes of different species, up to the level of complete spermatogenesis. This approach can be applied successfully to generate live progeny using sperm extracted from grafts originating from testes of newborn donors. The sperm are capable of supporting normal development and producing fertile male and female offspring after intracytoplasmic injection into mouse oocytes and embryo transfer into surrogate mothers. The grafted tissue was also capable of significantly normalizing reproductive hormone levels in the castrated recipients. This technique presents new avenues for experimentation. The recipient mouse can be regarded as a living incubator and a culture system of testicular tissue, allowing the experimental manipulation of several aspects of testis development and spermatogenesis. The successful generation of pups indicates that this technique can be used to study the testicular phenotype and to breed mutant or transgenic mouse strains with lethal postnatal phenotypes. The ability to generate sperm from the germ line ex vivo also paves the way for the development of new strategies for preserving fertility in boys undergoing cancer therapy.     

Comment identifier le spermatozoïde vivant?

Testicular sperm extraction (TESE) has been used to retrieve spermatozoa in patients with secretory azoospermia for intracytoplasmic sperm injection (ICSI). However, testicular spermatozoa have poor motility that significantly decreases after cryopreservation and thawing. The major difficulty with testicular spermatozoa is to distinguish between living and dead spermatozoa, as most spermatozoa are immotile. The aim of this study was firstly to report the various methods used to explore spermatozoa vitality. Most tests assess the functional and structural integrity of the sperm membrane, such as staining methods and hypo-osmotic swelling test (HOS-test). We then evaluates the potential of pentoxifylline (PTX), a phosphodiesterase inhibitor of the methylxanthine group, to improve the distinction between living and dead immotile testicular spermatozoa by increasing the number of post-thawed motile spermatozoa. We also analysed the results of 100 ICSI cycles performed with frozen-thawed testicular (n=72) and epididymal (n=28) spermatozoa treated with 3.5 mM PTX. To test the effect of PTX on motility, 14 samples of frozen-thawed testicular spermatozoa from eight patients with secretory azoospermia and six patients with excretory azoospermia were divided into three equal samples: one sample treated with 3.5 mM PTX, one sample initially migrated on two-layer Percoll gradient and then divided into two aliquots (one treated with 3.5 mM PTX, one without treatment), and the last sample without migration and without PTX treatment. The number of motile spermatozoa was evaluated in 10 μL of each sample with an inverted microscope at 15, 30, 60, 120 minutes and 24 hours. We also compared the outcome of ICSI in 100 cycles using frozen-thawed epididymal or testicular spermatozoa between secretory and excretory patients. PTX significantly increased the number of motile frozen-thawed testicular spermatozoa in secretory and excretory azoospermia. In excretory azoospermia, the number of motile spermatozoa was further increased when PTX was associated with migration on Percoll gradient, while PTX alone gave the best results in secretory azoospermia. Fertilization and pregnancy rates as well as embryo quality and division stages were comparable in the two groups. By increasing the number of motile frozen-thawed testicular spermatozoa, PTX improves the selection of living spermatozoa.