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Investigate in what extent sperm transcriptome of infertile men is different from that of fertile individuals.Semen samples were collected for determination of sperm parameters as well as for RNA isolation. Gene expression profile was investigated in spermatozoa of 8 infertile and 3 fertile men by microarray analysis using the Affymetrix Chip HG-U133 Plus 2.0.We observed up to 33-fold reduction expression of genes involved in spermatogenesis and sperm motility. Furthermore, there is an important decrease in expression of genes involved in DNA repair as well as oxidative stress regulation. In this study, we also show a striking drop in expression of histone modification genes.We found that transcription profile in germ cells of men with idiopathic infertility is different from that of fertile individuals. Interestingly, about 15% of the regulated genes (Eddy Rev Reprod 4:23-30, 1999) play a role in spermatogenesis.

SummaryThe aim of this study was to evaluate the advantages of the two-step embryo transfer (ET) strategy combining a day 2/3 ET with a day 5/6 blastocyst transfer. In an observational comparative study, 400 infertile women were enrolled from two assisted reproductive technology (ART) units according to inclusion criteria: age below 42 years and at least three embryos obtained on day 2 thus allowing an extended in vitro culture. Two groups were defined according to the ET strategy adopted: group 1 had a two-step ET; and group 2 had a day 2/3 ET with (subgroup 2a) or without (subgroup 2b) blastocysts cryopreserved on day 5/6. Live birth rate was significantly higher in group 1 than in subgroups 2a and 2b (36.5% versus 29.4% and 13.4%, respectively; p < 10−3). Multiple pregnancy rates were comparable between groups. After adjusting on major prognostic factors, the two-step ET strategy was still associated with a significantly higher live birth rate than the day 2/3 ET (OR = 2.23; 95% CI: 1.32–3.77). The two-step ET provides better live birth rates than the cleavage-stage ET. It does not increase multiple pregnancy rates if the number of embryos transferred is limited. It also prevents cycle loss when embryos fail to develop into blastocysts.

Résumé Introduction L’analyse fine de la morphologie des spermatozoïdes à un grossissement de 6600 x, appelée MSOME (motile sperm organellar morphology examination) et appliquée en ICSI, a donné naissance à l’IMSI (intracytoplasmic morphologically selected sperm injection). Cette technique est proposée aux couples en échecs répétés d’implantation embryonnaire en ICSI, dans le but d’obtenir une grossesse évolutive. Matériel et méthodes L’étude concerne une cohorte observationnelle de 11535 ICSI pratiquées avec du sperme éjaculé frais, de janvier 2004 à juillet 2009. Parmi celles-ci, 2509 ont été réalisées avec IMSI. Les paramètres étudiés sont: le taux de clivage à J2 par ovocyte injecté, le taux de grossesses cliniques par ponction et le taux de fausses couches spontanées, en fonction du rang de la tentative et/ou de la qualité du sperme du bilan. Ces données ont été comparées entre l’ICSI et l’IMSI et les comparaisons ont été faites à l’aide de Chi2 et d’analyse de variance. Résultats Nous n’avons pas observé de différence significative entre l’ICSI et l’IMSI en termes de taux de clivage à J2 et taux de grossesses cliniques. Par contre, le taux de grossesses, en IMSI de rang 1, est significativement plus élevé en cas d’anomalie du sperme si on regroupe les tératozoospermies avec les oligozoospermies et les oligotératozoospermies (34,4 vs 27,1%, p = 0,02). De plus, si l’on regroupe les tératozoospermies et les oligotératozoospermies, le taux de fausses couches est plus faible en IMSI comparé à l’ICSI, de manière proche de la significativité (12,6% vs. 19,6%, p = 0,08). Conclusion En présence d’une tératozoospermie sévère, l’IMSI semble augmenter les taux de grossesses et diminuer les taux de fausses couches.

Resume Objectifs Plusieurs etudes ont indique un declin relatif dans le temps de la qualite du sperme. Dans ce contexte, 101 404 spermogrammes-spermocytogrammes, executes dans un seul laboratoire de janvier 2000 a decembre 2009, ont ete etudies retrospectivement. Les variations des parametres du sperme ont ete analysees au cours de ces dix annees. Patients et methodes Pour chaque echantillon de sperme, l’âge, la duree de l’abstinence sexuelle, le volume, pH, la concentration des spermatozoides, le pourcentage de spermatozoides mobiles, atypiques (David modifie) et l’amplitude de deplacement lateral de la tete (ALH) ont ete analyses. Pour chaque parametre, la valeur moyenne par an a ete determinee. Les tests statistiques etaient des analyses de variance et l’etablissement d’un coefficient de correlation. Resultats Les donnees ont montre une augmentation de l’âge des patients tandis que la concentration spermatique est passee de 63,5 millions/mL en 2000 a 63,6 millions/mL en 2009 avec une legere chute de 2001 a 2008. La vitalite a augmente de 67,8 % en 2000 a 73,5 % de 2009 ainsi que la mobilite globale (de 37,7 % a 39,2 %) et progressive (de 34,6 % a 36,2 %). Le pourcentage des spermatozoides atypiques est passe de 64,5 % a 84,2 % (r = 0,43, p Discussion et conclusion Contrairement a de nombreux travaux, notre etude d’une tres grande serie d’analyses de spermes n’a pas montre de reel declin des parametres du sperme selon l’OMS au cours d’une periode de dix ans, excepte pour le pourcentage de spermatozoides atypiques, lequel a considerablement augmente au cours des annees.

Objective To investigate whether homocysteine, a well known inhibitor of methylation, which is produced after imprinting and other methylation processes, can be recycled to methionine in the oocyte, at least until the stage of maternal to zygotic transition (i.e., four- to eight-cell stage); before this stage, most of the biochemical processes are carried out with the use of maternal stores of protein and mRNA. Design A first approach using microarrays and then reverse-transcription polymerase chain reaction (RT-PCR) for methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase [MTR]), betaine-homocysteine methyltransferase (BHMT), and cystathionine-beta synthase (CBS). Setting Two private hospitals. Patient(s) Patients involved in IVF/ICSI procedures. Intervention(s) Germinal vesicle oocytes collected at the time of oocyte retrieval, RNA extraction amplification, RT-PCR, microarrays. Main Outcome Measure(s) mRNA expression of all the enzymes involved in the chain of methylation and recycling of homocysteine to methionine. Result(s) All of the enzymes required for methylation are present in the oocyte. Homocysteine can be recycled with BHMT and MTR. Conclusion(s) The human oocyte is able to regulate its Hcy level via remethylation using MTR and BHMT but not CBS. This aspect is important, because recent studies have shown that controlled ovarian hyperstimulation affects the homocysteine concentration in follicular fluid. This may regulate, at least in part, the risk of imprinting problems during IVF procedures.

Resume L’analyse morphologique du spermatozoide (ou spermocytogramme) fait partie du bilan d’infertilite du couple et permet de definir la normalite d’un sperme ; cependant, la relation entre morphologie spermatique et fertilite est loin d’etre etablie. Bien que plusieurs etudes ne montrent pas de relation entre formes anormales et resultats de l’ICSI, il semble neanmoins que les taux de succes dependent de l’aspect du spermatozoide injecte. L’examen de la morphologie fine du spermatozoide (et notamment de la tete), a tres fort grossissement (de × 6600 a × 12500) (MSOME) en temps reel, permet de selectionner les plus normaux et de les injecter (IMSI). Cette selection permet d’augmenter, dans certaines situations, les taux de grossesse evolutive et d’implantation. Des criteres ultramorphologiques ont ete etablis et il semble que le plus predictif de la qualite du spermatozoide soit la presence de vacuoles dans la tete du spermatozoide. De plus, ces vacuoles semblent reliees a des alterations de l’ADN (fragmentation et/ou decondensation) ayant pour consequence des defauts du developpement embryonnaire. Pour objectiver les observations, beaucoup d’auteurs ont essaye de mettre au point des classifications et d’etablir un spermocytogramme a fort grossissement qui pourrait etre utilisable en routine et pourquoi pas dans l’avenir, remplacer le spermocytogramme.

ICSI failures (essentially fertilization and implantation failures) can be due to the quality of the oocytes, the spermatozoon injected and its manipulation, uterine factors and paternal factors, such as DNA fragmentation of the spermatozoa. Pre-selection of the spermatozoon is the crucial phase for ensuring successful ICSI. The characteristics of the sperm must be checked under the microscope at a magnification of x 6600 as certain abnormalities, in particular nuclear vacuoles, cannot be seen at x 300. In our centre, we conducted a study on 72 patients which showed that with more than 30% of fragmented DNA, neither implantation or birth was achieved using ICSI. Performing IMSI on the same patients allowed us to obtain implantation rates of 17.4% with 30 to 40 % of fragmented DNA and of 33.3% above 40%. Births were achieved in 17.4% and 28.6% of cases respectively. IMSI has the advantage of allowing extremely careful selection of the spermatozoon microinjected, so that it is of normal shape, with the least vacuoles possible and a low DNA fragmentation rate. It is, however, a complicated technique that cannot be routinely performed.

In vitro maturation of human oocytes at the germinal vesicle (GV) stage could offer an alternative in several cases of female infertility. It however rests on a better knowledge of the quality of human oocyte. Using fluorescence imaging of DNA and of the transcription sites, combined with electron microscopy, we show that human oocytes follow size-dependent changes in chromatin configuration, transcription sites distribution and nuclear ultrastructure that follow those observed in mouse GV oocytes. We thus analyzed in mouse GV oocytes the phosphorylation dependence of the transcriptional activity. We show by Western blot that, while active GV oocytes have approximately the same proportion of hypo- and hyperphosphorylated forms of the RNA polymerase II (RNAP II), the hyperphosphorylated form is almost absent from inactive oocytes. We also show that (1) RNAP II-dependent transcription is much less sensitive to various kinase inhibitors in mouse oocytes than in somatic cells or mouse one-cell embryos, although the phosphorylation equilibrium of RNAP II was largely shifted towards the hypo-phosphorylated form upon treatment with these inhibitors (2) RNAP I is completely insensitive to kinase inhibitors in GV oocytes.

Embryos are conventionally selected for transfer based on the evaluation of the cleavage speed and extent of blastomere fragmentation. Here we examined whether the predictive value of these criteria, as indicators of the chance of embryo implantation, can be further potentiated by adding previously described criteria reflecting the regularity of pronuclear development. In a group of embryos selected for transfer in 380 fresh embryo transfer cycles according to the conventional criteria, the transfer of only those embryos that developed from zygotes judged normal at the pronuclear stage (pattern 0) gave significantly higher pregnancy (44.8%) and implantation (30.2%) rates compared with the pregnancy (22.1%; P < 0. 05) and implantation rates (11.2%; P < 0.001) for the transfers of only those embryos that developed from zygotes judged abnormal (non-pattern 0). The transfer of only one pattern 0 embryo was sufficient for the optimal chance of pregnancy (no differences in pregnancy rates after transfer of one, two or three pattern 0 embryos), whereas the transfer of two pattern 0 embryos mostly resulted in a twin pregnancy. The inclusion of the criteria based on pronuclear morphology can thus lead to the application of a single embryo transfer policy and optimize the selection of embryos for transfer and cryopreservation.

SummaryThe recently reported human pregnancies and births after fertilising oocytes with round spermatids recovered from the ejaculate of men with non-obstructive azoospermia have underscored the need for a more accurate evaluation of the nuclear and cytoplasmic maturation status of ejaculated germ cells. In this study we describe our first experience with a method combining the immunocytochemical visualisation of proacrosin with autosomal DNA fluorescencein situhybridisation (FISH) to assess ejaculated germ cells from patients with a spermiogenesis defect. The proacrosin immunoreactivity, analysed with the use of the monoclonal antibody 4D4, has been detected in cells of round spermatid size presenting a haploid FISH figure as well as in larger cells whose ploidy corresponds to primary and secondary spermatocytes. These observations are in agreement with previously published results obtained, with the use of the same antibody, by immunocytochemical analysis of histological sections of testicular tissue. All the cells of round spermatid size possessing proacrosin immunoreactivity were found to be haploid by FISH. On the other hand, some of the haploid cells of round spermatid size did not possess proacrosin immunoreactivity. The structural pattern of proacrosin immunoreactivity was highly variable both in spermatids and in younger spermatogenic cells. These data show that cell size is the main criterion to be used for the identification of ejaculated round spermatids, whereas the presence of the developing acrosome represents only an auxiliary criterion. The scoring of acrosomal development in ejaculated spermatids may be useful as part of pre-treatment diagnosis before the inclusion of infertile couples in a spermatid conception programme.

Objective To characterize a potential genetic cause for methylation errors described in oligozoospermia. Design Analysis of PEG1/MEST -DMR and H19 -DMR methylation level in sperm, in parallel with the study of several genes on the Y chromosome, DNMT3A , and DNMT3L . Clinical outcome was also looked at regarding PEG1/MEST -DMR and H19 -DMR methylation level in sperm. Setting Research and diagnostic laboratories. Patient(s) One hundred nineteen normospermic and 175 oligozoospermic men consulting for couple infertility. Intervention(s) We studied PEG1/MEST -DMR and H19 -DMR methylation profiles in 294 men. We searched for Y chromosome gene aberrations and for mutations in both DNMT3A and DNMT3L genes in men showing epimutations. Assisted reproductive technology (ART) outcomes were also investigated. Main Outcome Measure(s) Sperm samples were collected from 294 volunteers for genomic DNA isolation that was used to study methylation profiles in imprinted loci and Y chromosome SMCY , DNMT3A , and DNMT3L genes. Pregnancy rate was also studied after ART treatment using sperm showing epimutations. Result(s) Epimutations in H19 -DMR and PEG1/MEST -DMR were found in 20% and 3% of oligozoospermic men, respectively. We identified an amino acid change in DNMT3A in one case and in DNMT3L in eight men with altered methylation profiles. No mutations were detected in SMCY or in selected Y chromsome genes. No correlation between ART outcome and epimutations was found. Conclusion(s) We observed epimethylations in spermatozoa of oligozoospermic individuals, but no association was found with genetic variants or in the ART outcome.

La culture prolongee correspondait au depart a une necessite : les biotechnologies chez les animaux domestiques a interet zootechnique necessitent, pour le transfert embryonnaire, un transfert au stade blastocyste. En effet, le transfert aux stades precoces, apres fecondation in vitro et culture de courte duree, n’est tout simplement pas possible : les embryons sont expulses de l’uterus. Seul le transfert de morula tardives (5 jours) ou de blastocyste est operant. Les problemes de culture de l’embryon du stade 1 au blastocyste se heurtent, dans de nombreux cas, a un blocage au moment du cycle cellulaire correspondant a la transition du controle maternel au controle embryonnaire (MZT ou maternal to zygotic transition) : souris : 2–4 C, homme : 4–8 C, vache, lapin : 8–16 C. Ce cycle cellulaire dure en moyenne 24 heures pendant lesquelles demarrent les premieres transcriptions embryonnaires ; l’embryon prend son autonomie par rapport aux reserves d’ARN et de proteines stockees pendant la croissance de l’ovocyte, qui se sont epuisees pendant les premieres divisions.

Depuis quelques annees, plusieurs publications ont mis en exergue l’avantage de l’examen du sperme au fort grossissement (× 6 600), permettant l’evaluation de la morphologie du noyau spermatique et la densite de sa chromatine en temps reel avant micro-injection ovocytaire. Ce procede est communement appele motile sperm organelle morphology examination (MSOME). La technique d’ICSI est appelee intracytoplasmic morphologically selected sperm injection (IMSI). En reproduction, nous deplorons helas plus d’echecs que de succes. Les causes des echecs sont multiples, d’origine maternelle mais aussi paternelle. En effet, un sperme inadequat peut conduire au pire a un defaut de fecondation ou de developpement embryonnaire precoce et au mieux a une implantation embryonnaire suivie d’une fausse couche.

Objective To investigate the relationship between MTHFR and MTRR genetic variants with respect to both blood plasma homocysteine concentration and sperm counts. Design Polymerase chain reaction followed by specific enzymatic digestion to determine the genotype of the individuals and blood plasma homocysteine quantification by high-performance liquid chromatography. Setting Research laboratory. Patient(s) Two hundred sixty-eight men seeking infertility counseling and 254 partners of infertile women. Intervention(s) We studied three MTHFR (c.1286A→C, c.665C→T and c.203G→A) and two MTRR (c.66A→G and c.524C→T) single-nucleotide polymorphisms and characterized sperm parameters in both oligozoospermic and normospermic men. A cohort of 522 men was examined for this study. A subgroup of 103 men was constituted for quantification of Hcy levels. Main Outcome Measure(s) Semen samples were collected for determinations of sperm concentration, motility, and morphology according to World Health Organization guidelines as well as for DNA isolation. Blood samples of the corresponding individuals were obtained to quantify plasma homocysteine levels. Result(s) We did not observe a relationship between homocysteinemia and sperm counts. The MTHFR c.665C→T variant is associated with mild hyperhomocysteinemia in blood plasma in the TT homozygous state. Conclusion(s) No association was found between MTHFR / MTRR genetic variants and sperm counts. Although no association was observed with reduced sperm counts, the MTHFR 665TT genotype is associated with a significant increase in blood plasma homocysteine levels.

The effect of paternal age on sperm DNA fragmentation and decondensation was determined in a retrospective study involving 1769 patients. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay was used to assess fragmentation, and DNA decondensation was measured with either chromomycin or aniline blue staining. The impact of atypical forms was also analysed. DNA fragmentation increases with age, but is independent of the percentage of atypical forms. Both staining techniques revealed a negative correlation between the quality of sperm packaging and the percentage of atypical forms. Decondensation increases with increasing age and fragmentation when measured with chromomycin; however, an inverse relationship is observed when testing is performed using aniline blue. These observations are discussed in relation to the specificity of the dyes, the deposition of protamines and the impact of age and reactive oxygen species on protamine cross-linking.

In a preliminary, unpublished randomized study conducted in 2000 on 39 patients, including a placebo group, it was observed that the addition of growth hormone (GH) during ovarian stimulation in patients with poor-quality oocytes increased the pregnancy rate. However, the results were not statistically significant due to the small number of patients in each group. A protocol with 8 IU GH was tested in 291 patients with three or more previous failures of embryo transfer for no clearly identifiable reasons. The analysis was restricted to patients receiving either recombinant FSH or human menopausal gonadotrophin (HMG) (n = 245). They were compared retrospectively to all patients with three or more failures during the same period of time but stimulated only with recombinant FSH or HMG, without GH, in an observational study design. Co-stimulation with GH gave better results in terms of number of oocytes collected and embryos obtained. Pregnancy rate per retrieval was higher than in the control group (25.7% versus 18.2%, P < 0.01) and reached a level similar to the one observed in the study centre for the whole population. Ovarian stimulation associated with GH can be proposed for patients with a history of repeated assisted reproduction failures. An improvement of cytoplasmic competence is proposed as an explanation.

More than 17,000 intrauterine insemination (IUI) cycles were analysed retrospectively with respect to outcome according to differing aetiologies of infertility. The quantity and motility of spermatozoa in the final preparation used for insemination had a positive effect on the outcome, as classically observed in the past. It was found that advanced maternal age had a negative effect on the pregnancy rate and was associated with increased miscarriage rate. More interestingly, an exactly parallel effect was found for paternal age. The impact of increased age on necrospermia and sperm DNA structure is discussed as a probable direct cause of this paternal effect.

Objective To investigate DNA fragmentation by using terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling in relation to World Health Organization parameters and computer-aided sperm analysis (CASA) in sperm to determine the possibility of obtaining a correlation among CASA parameters, sperm morphology, and DNA fragmentation. Design Sperm analysis according to World Health Organization parameters, terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) for sperm DNA fragmentation, and CASA for sperm movement. Prospective study. Setting All the patients were under clinical management, consulting for hypofertility at a fertility center in France. Patient(s) One thousand six hundred thirty-three men who were referred for infertility investigation, including a complete sperm analysis. Intervention(s) Sperm analysis and DNA damage testing. Main Outcome Measure(s) Sperm morphology, DNA fragmentation, and movement characteristics. Result(s) One third of the patients had a TUNEL rate of >30%. Analysis of the 21 semen parameters tested revealed that 7 of them were significantly correlated with the TUNEL results. Conclusion(s) World Health Organization sperm parameters and DNA damage are complementary, rather than strongly linked. This should be considered to more fully understand the paternal contribution in assisted reproductive technologies failures.

Reactive oxygen species (ROS) have a negative impact on sperm DNA, leading to the formation of oxidative products such as 8-oxo-7,8-dihydroxyguanosine. This compound causes fragmentation and, thus, has a mutagenic effect. Patient treatment with oral antioxidant vitamins is, therefore, standard practice for male infertility, in an attempt to decrease formation of ROS and improve fertility. In this study, the DNA fragmentation index and the degree of sperm decondensation were measured using the sperm chromatin structure assay before and after 90 days treatment with antioxidant vitamins associated with zinc and selenium. Antioxidant treatment led to a decrease in sperm DNA fragmentation (–19.1%, P < 0.0004), suggesting that at least part of the decay was linked to ROS. However, it also led to an unexpected negative effect: an increase in sperm decondensation with the same order of magnitude (+22.8%, P < 0.0009). The opening of interchain disulphide bridges in protamines may explain this aspect, as antioxidant vitamins, especially vitamin C, are able to open the cystin net, thus interfering with paternal gene activity during preimplantation development. This observation might explain the discrepancy observed concerning the role of these antioxidant treatments in improving male fertility.

Previous studies have shown that repeated intracytoplasmic sperm injection (ICSI) failures can be caused by a paternal effect. Other studies have suggested that ICSI results are compromised if morphologically abnormal spermatozoa are injected into oocytes. This study was undertaken to evaluate the usefulness of a high-magnification optical system to select spermatozoa to be used for ICSI (high-magnification ICSI) in couples with repeated conventional ICSI failures. Couples with two or more previous conventional ICSI failures underwent an additional conventional ICSI attempt, followed by a high-magnification ICSI attempt. The outcomes of the two sequential attempts were compared. In 72 of these patients, sperm DNA integrity was assessed. In the whole group of 125 couples with repeated ICSI failures, high-magnification ICSI improved clinical outcomes (pregnancy, implantation, delivery and birth rates) without affecting biological outcomes (fertilization and cleavage rates, embryo morphology). The improvement of clinical ICSI outcomes was evident both in patients with an elevated degree of sperm DNA fragmentation and in those with normal sperm DNA status. It is concluded that high-magnification ICSI improves clinical outcomes in couples with previous repeated conventional ICSI failures.

Objective To test the hypothesis that the concentration of early follicular phase serum antimullerian hormone (AMH) or mullerian-inhibiting substance (MIS) is a useful marker of ovarian response and assisted reproductive technology (ART) outcome. Design Retrospective analysis of day 3 serum samples drawn before treatment. Setting Private ART program. Patient(s) One hundred nine consecutive serum samples from women younger than 42 years of age who were undergoing ovulation induction for IVF. Intervention(s) Follicular aspiration for IVF after ovarian stimulation with FSH in a down-regulated cycle using GnRH-a treatment. Main outcome measure(s) Correlations between day 3 serum AMH/MIS, E2, FSH, inhibin B levels, and IVF outcome (i.e., number of retrieved mature oocytes, number and quality of embryos obtained, ongoing clinical pregnancy rates). Multivariate regression analysis on categorical data was performed to describe a predictive model of clinical pregnancy outcome. Result(s) Mean serum AMH/MIS value for clinical pregnancy (n = 38) was 2.4 ng/mL, in comparison to 1.1 ng/mL for those who did not become pregnant (n = 71). No differences were noted in mean values for day 3 FSH, inhibin B, or E2 between groups. Multivariate regression analysis demonstrated that day 3 serum AMH/MIS had the greatest independent contribution in predicting pregnancy outcomes. Conclusion(s) These data demonstrate a strong association between day 3 serum AMH/MIS level and IVF outcome in women younger than 42 years of age. Higher AMH/MIS concentrations are associated with a greater number of mature oocytes, a greater number of embryos, and ultimately a higher clinical pregnancy rate. Furthermore, AMH/MIS may offer greater prognostic value than other currently available serum markers of ART outcome.

Background Using fluorescence imaging, an a posteriori multiparametric analysis was performed of human oocytes which failed to give pronucleated zygotes after IVF in cases of very low rates of fertilization or complete fertilization failure. Methods The analysis included: (i) the state of the maternal and paternal chromatin; (ii) quality of the metaphase II oocytes; and (iii) cortical granule (CG) distribution. Results Most oocytes were arrested in metaphase II, but they were abnormal in 50% of cases. The incidence of spindle and chromosome aberrations was strongly influenced by maternal age (69% for 40- to 45-year-old women versus 35% for 26- to 33-year-olds), and sperm chromatin was always condensed in immature oocytes, and fully decondensed only in normal metaphase II. The migration of CGs appeared to be associated with achievement of nuclear maturation at the time of puncture. Conclusions These factors, when analysed on a complete set of oocytes from the same patient, provided information about potential causes of IVF failure, and also represented part of an 'oocyte quality evaluation' to select the assisted fertilization technique most suitable for each patient. For example, when the majority of oocytes were judged non-fertilizable at a first attempt, no pregnancy was registered at any subsequent attempt.

Since the conception of in vitro fertilization (IVF), it has been gradually accepted that artificial reproductive technology does not imply a higher risk of congenital malformation.

Intracytoplasmic sperm injection (ICSI) represents spectacular progress in the treatment of male infertility. This technique makes the conception of a child possible in cases where only a few living spermatozoa (mostly motile ones) can be obtained from the ejaculate or recovered from the male genital tract. The intra-oocyte injection of spermatozoa that originates from the ejaculate, the epididymis, or the testis can yield fertilization rates (2 PN zygotes/ intact oocyte) of 62%, 65.6%, and 58%, respectively. The respective clinical pregnancy rates per transfer from the three sperm sources are 33.4%, 39.5%, and 35.8% (see Tables 13.1–13.4).

Germ cell apoptosis was evaluated in 11 men suffering from nonobstructive azoospermia and enrolled in a spermatid conception programme. In six of these patients, round spermatids (Sa stage) were the most advanced spermatogenic cells recovered from testicular biopsy samples. This condition is referred to as complete spermiogenesis failure. In the remaining five men, a few late elongated spermatids (Sd stage) were unexpectedly found in the testicular biopsy samples on the day of treatment. This condition is referred to as incomplete spermiogenesis failure. Germ cell apoptosis in both groups of patients was examined by analysing cell smears prepared from mechanically disintegrated testicular tissues using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), which detects apoptosis-specific DNA fragmentation, and annexin-V binding, detecting apoptosis-related translocation of plasma membrane phosphatidylserine to the membrane's outer surface. Both methods were combined, in double-fluorescence labelling preparations, with immunocytochemical detection of proacrosin, a specific germline marker. Patients with complete spermiogenesis failure had significantly higher frequencies of primary spermatocytes and round spermatids carrying the apoptosis-specific DNA damage in comparison with patients with incomplete spermiogenesis failure. Surprisingly, apoptosis-related phosphatidylserine externalization occurs rarely until the advanced stages of spermiogenesis. Since externalized phosphatidylserine is expected to be involved in the recognition of apoptotic cells by phagocytes, apoptotic spermatocytes and round spermatids may not be removed easily by phagocytosis. The high frequency of DNA damage in round spermatids from patients with complete spermiogenesis failure explains the low success rates of spermatid conception in these cases. The evaluation of apoptosis can help predict success rates of spermatid conception.

In-vitro differentiation of spermatogenic cells is a potential approach to the treatment of male sterility due to spermatogenic arrest. This is a pilot study evaluating meiotic, morphogenetic and cytoplasmic maturation of spermatogenic cells from 18 patients with obstructive azoospermia, during in-vitro culture of partly disintegrated testicular biopsy samples in the presence or absence of recombinant follicle stimulating hormone (rFSH). Meiotic progression was detectable only in the presence of rFSH in culture medium. FSH-dependent condensation, peripheral migration and protrusion of spermatid nuclei, together with FSH-independent flagellar growth, were the main events indicating post-meiotic sperm cell differentiation. rFSH also promoted the progression of spermatid cytoplasmic maturation, reflected by acceleration of acrosomal development. These differentiation events appeared to be mediated by humoral activity of Sertoli cells, without the need for a direct Sertoli-sperm cell contact. These findings provide a background for similar studies in patients with non-obstructive azoospermia. If reproducible in the latter group, transmeiotic in-vitro differentiation of primary spermatocytes may be useful in cases of complete maturation arrest, whereas the development of culture-specific forms may help select viable spermatids in cases of complete spermiogenesis failure.