Proteomic identification of target proteins in normal but nonfertilizing sperm.

OBJECTIVE: To identify the male molecular causes of failures of IVF (with a deficient binding of spermatozoa to the zona pellucida, without any obvious oocyte anomaly), which are undetected by classical sperm analysis. DESIGN: Case-control prospective study. SETTING: University hospital. PATIENT(S): Proteomic profiles of spermatozoa in patients with a complete failure of fertilization and no spermatozoa bound to the zona pellucida were compared with those of controls (men with normal fertilization and cleavage rates after classical IVF for tubal indication). INTERVENTION(S): All samples were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) after being divided into three fractions according to their isoelectric point. MAIN OUTCOME MEASURE(S): Differentially expressed proteins between infertile men and controls were identified by mass spectrometry. RESULT(S): Seventeen proteins differentially expressed between cases and controls were found. Twelve of these proteins were identified by mass spectrometry, and two may influence gametes interaction: laminin receptor LR67 and L-xylulose reductase (P34H). CONCLUSION(S): This study shows that 2D-DIGE might be useful in finding potential targets for diagnosis and prognosis of idiopathic infertility in IVF.

In Vitro fertilization failure of normozoospermic men: search for a lack of testicular isozyme of angiotensin-converting enzyme.

BACKGROUND: Angiotensin converting enzyme (ACE) is a metalloprotease with two isoforms. The somatic isoform is a key component of the renin-angiotensin system; its main function is to hydrolyse angiotensin I into angiotensin II. The germinal or testicular isoform (tACE) located at the plasma membrane of the spermatozoa, plays a crucial role in the spermatozoa-oocyte interaction during in vivo fertilization, in rodents. Disruption of the tACE in mice has revealed that homozygous male tACE-/- sire few pups despite mating normally. Few spermatozoa from these tACE-/- mice are bound to the zona pellucida (ZP) despite normal semen parameters. Based on these findings in mice models, we hypothesized that some infertile men that have the same phenotype as the tACE-/- mice, ie normal semen parameters and a lack of sperm bind to the ZP in vitro, may have a tACE defect. METHODS: Twenty four men participated to this study. The case subjects (n = 10) had normal semen parameters according to the WHO guidelines (WHO 1999) but a total in vitro fertilization failure with absence of sperm fixation to the ZP. The control subjects (n = 14) also had normal semen parameters and a normal fertilization rate ≥65%. We investigated the tACE expression in spermatozoa by Western-Blot and performed a DNA sequencing of the tACE gene. RESULTS: Three case-subjects and one control-subject had no tACE expression. There were no statistic differences between the two groups. No mutation was detected in the tACE DNA sequence. CONCLUSIONS: Our results didn't show any involvement of tACE in human fertilization especially in ZP binding.

INTRODUCTION: L'enzyme de conversion de l'angiotensine (ACE) est une métalloprotéase qui se présente sous deux isoformes. Une isoforme somatique qui intervient dans la régulation de la pression artérielle via le système rénine-angiotensine et une isoforme testiculaire spermatique (ACEt) qui interviendrait dans la fixation du spermatozoïde aux enveloppes ovocytaires (zone pellucide et/ou oelemme). Des expériences d'invalidation du gène de l'ACEt chez la souris ont montré que les mâles homozygotes ACEt−/− présentaient une faible descendance in vivo et une fixation faible des spermatozoïdes à la zone pellucide (ZP) in vitro et ce malgré un sperme normal. Ces résultats nous ont permis d'émettre l'hypothèse que certains échecs de fécondation en fécondation in vitro classique (FIV) chez des patients à sperme normal seraient en rapport avec un déficit de l'ACEt au niveau des spermatozoïdes. MATÉRIEL ET MÉTHODES: Vingt quatre patients ont été inclus dont 10 cas avec un sperme normal et un échec total de fécondation lors d'une FIV et 14 témoins ayant les mêmes caractéristiques spermatiques mais présentant de bons taux de fécondation (≥65%). L'étude de l'expression protéique de l'ACEt a été faite par western-blot sur les culots de spermatozoïdes. Un séquençage du gène codant pour l'ACEt a été fait sur l'ADN lymphocytaire. RÉSULTATS: Trois cas et un témoin présentaient un défaut d'expression de l'ACEt sans diffèrence statistiquement significative entre ces deux groupes. En PCR, aucune mutation n'a été retrouvée après séquençage du gène de l'ACEt. CONCLUSIONS: Nos résultats n'ont pas objectivé une implication de l'ACEt dans l'interaction entre ovocyte et spermatozoïde et notamment dans la fixation à la ZP.

Characterization of a recurrent poor-quality embryo morphology phenotype and zygote transfer as a rescue strategy.

Some patients in IVF programmes repeatedly display an abnormal embryonic development characterized as soon as day 2 post fertilization by a high rate (>60%) of highly fragmented embryos (⩾40% of cytoplasmic fragments) leading to recurrent IVF failures. This study postulated that, for various maternal reasons, some embryos were unable to withstand the in-vitro environment and an early pronucleate-stage transfer was proposed to these couples. Fifty-three patients with recurrent IVF failures (a mean of 2.8±1.0 previous attempts) characterized by low embryonic quality (a mean of 62.7% of the embryos with extended fragmentation) were included this transfer protocol. As in previous cycles, the mean number of oocytes retrieved and the fertilization rate were normal. The mean number of zygotes per transfer was 2.24. Fourteen clinical pregnancies were obtained, representing a pregnancy rate and a delivery rate per oocyte retrieval of 26.4% and 18.9%, respectively. Recurrent heavy and early embryo fragmentation in vitro characterizes around 3% of IVF couples and leads to lack of transfer or implantation failure. These data on fresh zygote transfers are encouraging and may provide a valid alternative solution for some of these patients. Some patients in IVF programmes repeatedly display an abnormal embryonic development characterized as soon as day 2 post fertilization by a high rate of highly fragmented embryos, leading to recurrent IVF failures. We hypothesized that, for various reasons, some embryos were unable to withstand the in-vitro environment and an early pronucleate stage transfer was proposed to these couples. Fifty-three patients with recurrent IVF failures characterized by low embryonic quality were included in this transfer protocol. As in previous cycles, the mean number of oocytes retrieved and the fertilization rate were normal. The mean number of zygotes per transfer was 2.24. Fourteen clinical pregnancies were obtained, representing a pregnancy rate and a delivery rate per oocyte retrieval of 26.4% and 18.9%, respectively. Recurrent early and heavy embryo fragmentation in vitro characterizes around 3% of IVF couples and leads to lack of transfer or implantation failure. Our data on fresh zygote transfers are encouraging and may provide a valid alternative solution for these patients.

Human male infertility associated with mutations in NR5A1 encoding steroidogenic factor 1.

One in seven couples worldwide are infertile, and male factor infertility accounts for approximately 30%-50% of these cases. Although many genes are known to be essential for gametogenesis, there are surprisingly few monogenic mutations that have been conclusively demonstrated to cause human spermatogenic failure. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis, and it is expressed in the steroidogenic tissue of the developing and adult human gonad. Mutations of NR5A1 have been reported in 46,XY disorders of sex development and in 46,XX primary ovarian insufficiency. To test the hypothesis that mutations in NR5A1 cause male infertility, we sequenced NR5A1 in 315 men with idiopathic spermatogenic failure. We identified seven men with severe spermatogenic failure who carried missense mutations in NR5A1. Functional studies indicated that these mutations impaired NR5A1 transactivational activity. We did not observe these mutations in more than 4000 control alleles, including the entire coding sequence of 359 normospermic men and 370 fertile male controls. NR5A1 mutations are found in approximately 4% of men with otherwise unexplained severe spermatogenic failure.

Progressive alcohol-induced sperm alterations leading to spermatogenic arrest, which was reversed after alcohol withdrawal.

This is a report of a 6-year follow-up of a male patient's semen parameters during heavy chronic alcohol intoxication and after withdrawal. A slowly progressive negative impact of alcohol could be observed: isolated moderate teratozoospermia was firstly noted followed by oligoasthenoteratospermia. Then a severe worsening resulted in cryptozoospermia and ultimately in azoospermia. At this moment, the histological analysis of a testicular biopsy revealed a maturation arrest of the germinal cells at the pachytene stage with no mature sperm cells. Alcohol withdrawal was then obtained, allowing a very fast and drastic improvement of semen characteristics; strictly normal semen parameters were observed after no more than 3 months. Taking into consideration these data, patients should be questioned about their alcohol intake before assisted reproductive technology and should be informed about this adverse effect. Moreover, this case report emphasizes how quickly benefits can be obtained after withdrawal, even in the case of heavy chronic alcohol intake.

Successful pregnancy by insemination of spermatozoa in a woman with a human seminal plasma allergy: should in vitro fertilization be considered first?

OBJECTIVE: To raise the possibility that pregnancy can be obtained by assisted reproductive techniques in patients with human seminal plasma allergy. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A woman consulted for a 3-year primary infertility. She reported lack of intercourse because of a seminal plasma allergy. INTERVENTION(S): One intrauterine insemination associated with antihistamine treatment was performed with carefully washed spermatozoa. RESULT(S): Immediately after the insemination, the patient had an allergic reaction treated by steroids. Fortunately, this single attempt led to a successful pregnancy, and the patient gave birth to a healthy girl. CONCLUSION(S): Pregnancies can be obtained in patients with seminal plasma hypersensitivity by means of intrauterine insemination. However, serious complications may occur after performing intrauterine insemination, which is not a totally reliable method to prevent an allergic reaction. Thus a multidisciplinary team should follow such patients carefully and watch for the potential risks and side effects. In vitro fertilization may represent the more cautious option.

Determinants of pregnancy rate in the donor oocyte model: a multivariate analysis of 450 frozen-thawed embryo transfers.

BACKGROUND: Conflicting results have been published about the determinants of pregnancy after oocyte donation (OD). We used the OD model to determine predictive factors of pregnancy in the recipient after frozen-thawed embryo transfer (FTET) in a specific series where all the embryos were cryopreserved without any prior selection for fresh transfer. METHODS: We report a retrospective study in a university tertiary care center. Multivariate analysis and logistic regression were used to identify predictive factors of pregnancy in a series of 450 OD FTET cycles in 198 infertile women between January 1992 and December 2006. RESULTS: The mean (+/-SD) recipient age was 35.7 (+/-4.5). Impaired ovarian function was the main indication for OD. The mean +/- SD (range) number of embryos transferred was 1.65 +/- 0.5 (1-3). Overall clinical pregnancy, implantation and delivery rates were 30, 18 and 23%, respectively. After univariate analysis, pregnancy rates were significantly higher in recipients under 35 years, in women with a body mass index (BMI) <30 kg/m(2), in women with an endometrial thickness of > or =8 mm, in amenorrheic women and in women not receiving pituitary down-regulation before endometrial preparation. Using multivariate analysis, the BMI, endometrial thickness and the use of pituitary down-regulation were independent predictors of pregnancy, regardless of age. CONCLUSIONS: This study supports that endometrial thickness of <8 mm, obesity and the use of GnRH analogue pituitary down-regulation before endometrial priming negatively impact pregnancy rates, independently of the recipient's age.

Lack of association between genetic polymorphisms in enzymes associated with folate metabolism and unexplained reduced sperm counts.

BACKGROUND: The metabolic pathway of folate is thought to influence DNA stability either by inducing single/double stranded breaks or by producing low levels of S-adenosyl-methionine leading to abnormal gene expression and chromosome segregation. Polymorphisms in the genes encoding enzymes in the folate metabolism pathway show distinct geographic and/or ethnic variations and in some cases have been linked to disease. Notably, the gene Methylenetetrahydrofolate reductase (MTHFR) in which the homozygous (TT) state of the polymorphism c.665C>T (p.A222V) is associated with reduced specific activity and increased thermolability of the enzyme causing mild hyperhomocysteinemia. Recently several studies have suggested that men carrying this polymorphism may be at increased risk to develop infertility. METHODOLOGY/PRINCIPAL FINDINGS: We have tested this hypothesis in a case/control study of ethnic French individuals. We examined the incidence of polymorphisms in the genes MTHFR (R68Q, A222V and E429A), Methionine synthase reductase MTRR; (I22M and S175L) and Cystathionine beta-synthase (CBS; G307S). The case population consisted of DNA samples from men with unexplained azoospermia (n = 70) or oligozoospermia (n = 182) and the control population consisted of normospermic and fertile men (n = 114). We found no evidence of an association between the incidence of any of these variants and reduced sperm counts. In addition haplotype analysis did not reveal differences between the case and control populations. CONCLUSIONS/SIGNIFICANCE: We could find no evidence for an association between reduced sperm counts and polymorphisms in enzymes involved in folate metabolism in the French population.

Learning curve of vitrification assessed by cumulative summation test for learning curve (LC-CUSUM).

We used the cumulative summation test for learning curve (LC-CUSUM), a specifically designed statistical tool, to evaluate the first 50 procedures performed by a trainee in vitrification and to provide a usable model for monitoring the learning process of this technique. Given the lack of models to evaluate IVF technologies, the CUSUM methodology could prove useful for quality control in laboratories.

Mutational analysis of the WNT gene family in women with Mayer-Rokitansky-Kuster-Hauser syndrome.

The aim of this study is to determine if Müllerian agenesis has a genetic basis linked to the WNT genes. Genomic DNA analyses for mutations in the coding sequences of four members of this family in a series of 11 women with Mayer-Rokitansky-Kuster-Hauser syndrome found four variants in the coding sequence of these genes, but causal mutations were not observed. This supports the hypothesis that mutations in the coding sequence of WNT4, WNT5A, WNT7A, and WNT9B are not responsible for the Mayer-Rokitansky-Kuster-Hauser syndrome.

Y-chromosome AZFc structural architecture and relationship to male fertility.

OBJECTIVE: To determine if there is a relationship between various forms of partial AZFc deletions and spermatogenic failure. DESIGN: Case-control study. SETTING: Infertility clinic (Tenon Hospital, Paris). PATIENT(S): 557 men, comprising 364 infertile men from mixed ethnic backgrounds, and 193 men with known fertility (n = 84) and/or normospermic (n = 109). INTERVENTION(S): Characterization of 32 partial AZFc deletions. MAIN OUTCOME MEASURE(S): DAZ gene cluster divided into two families (DAZ1/2 and DAZ3/4), CDY1 gene, and Y-chromosome haplogroups. RESULT(S): We observed 18 partial AZFc deletions in 364 (4.95%) infertile men compared with 14 out of 193 (7.25%) in the control normospermic/fertile group. CONCLUSION(S): The analysis of informative Y-chromosome single nucleotide variants combined with Y-chromosome haplogroup definition enabled us to infer seven deletion classes that occur on a minimum of six Y-chromosome parental architectures. We found no relationship between either the presence or the absence of DAZ1/2, DAZ3/4, CDY1a, or CDY1b with spermatogenic failure at least on one Y-chromosome lineage. The DAZ dosage and Southern blot analyses indicated that the majority of individuals tested carried two copies of the DAZ gene, indicating a partial AZFc deletion. Our data are consistent with the hypothesis that, at least in our study populations, partial AZFc deletions may have a limited impact on fertility.

[Y chromosome and male infertility: what is a normal Y chromosome?]. / Chromosome Y et infertilité masculine: qu'est-ce qu'un chromosome Y normal?

The human Y chromosome contains a number of genes and gene families that are essential for germ cell development and maintenance. Many of these genes are located in highly repetitive elements that are subject to rearrangements. Deletion of azoospermia factor (AZF) regions AZFa, AZFb, and AZFc are found in approximately 10-15% of men with severe forms of spermatogenic failure. Several partial AZFc deletions have been described. One of these, which removes around half of all the genes within the AZFc region, appears to be present as an inconsequential polymorphism in populations of northern Eurasia. A second deletion, termed gr/gr, also results in the absence of several AZFc genes and it may be a genetic risk factor for spermatogenic failure. However, the link between these partial deletions and fertility is unclear. The gr/gr deletion is not a single deletion but a combination of deletions that vary in size and complexity and result in the absence of different genes. There are also regional or ethnic differences in the frequency of gr/gr deletions. In some Y-chromosome lineages, these deletions appear to be fixed and may have little influence on spermatogenesis. Most of these data (gene content and Y chromosome structure) have been deduced from the reference Y chromosome sequence deposited in NCBI. However, recently there have been attempts to define these types of structural rearrangements in the general population. These have highlighted the considerable degree of structural diversity that exist. Trying to correlate these changes with the phenotypic variability is a major challenge and it is likely that there will not be a single reference (or normal) Y chromosome sequence but many.

Influence of sickle cell disease and treatment with hydroxyurea on sperm parameters and fertility of human males.

BACKGROUND: Recent progress in the treatment of sickle cell disease, in particular the use of hydroxyurea, has considerably modified the prognosis of this disease. Many more patients now reach reproductive age. The objective of this study was to assess the potential impact of hydroxyurea on the semen of patients. DESIGN AND METHODS: In this retrospective multicenter study, we evaluated the sperm parameters and fertility of 44 patients and analyzed the potential impact of hydroxyurea. RESULTS: We report data from the largest series so far of semen analyses in patients with sickle cell disease: 108 samples were analyzed, of which 76 were collected before treatment. We found that at least one sperm parameter was abnormal in 91% of the patients before treatment, in agreement with published literature. All sperm parameters seemed to be affected in semen samples collected during hydroxyurea treatment, and this impairment occurred in less than 6 months, later reaching a plateau. Furthermore, after hydroxyurea cessation, while global results in 30 patients were not statistically different before and after hydroxyurea treatment, in four individuals follow-up sperm parameters did not seem to recover quickly and the total number of spermatozoa per ejaculate fell below the normal range in about half the cases. CONCLUSIONS: The observed alterations of semen parameters due to sickle cell disease seem to be exacerbated by hydroxyurea treatment. Until prospective studies reveal reassuring findings, we suggest that a pre-treatment sperm analysis be performed and sperm cryopreservation be offered to patients before hydroxyurea treatment.

Tail stump syndrome associated with chromosomal translocation in two brothers attempting intracytoplasmic sperm injection.

OBJECTIVE: To raise the possibility that a familial chromosomal translocation associated with teratozoospermia can disrupt a gene necessary for flagellum assembly. DESIGN: Case report. SETTING: University hospital. PATIENT(S): Two brothers with infertility related to anomalies of meiotic division and of the flagella assembly, presenting the same balanced 5-12 autosomal translocation. INTERVENTION(S): Several intracytoplasmic sperm injection (ICSI) cycles in our IVF department for both couples. MAIN OUTCOME MEASURE(S): Sperm analysis, karyotypes, electron microscopy, and fluorescence in situ hybridization (FISH) analysis of spermatozoa performed using probes coding for chromosomes X, Y, 13, 18, and 21. RESULT(S): In both brothers, sperm analysis indicated a tail stump syndrome. Electron microscopy analysis displayed complex abnormalities, which were probably related to meiotic errors. The FISH analysis indicated an increase of diploid germ cells. Karyotypes of both patients revealed the same balanced chromosomal t(5;12) (p15.1; q21) translocation. Results of ICSI cycles were comparable for both couples. A twin pregnancy was achieved in one of these two couples, but a spontaneous miscarriage occurred at 10 weeks of gestation. CONCLUSION(S): The flagella anomalies raise the possibility that the translocation disrupts a gene necessary for the flagellum assembly, although a mutation in a gene unrelated to the chromosome breakpoints cannot be excluded.

GR/GR deletions within the azoospermia factor c region on the Y chromosome might not be associated with spermatogenic failure.

No statistically significant difference in deletion frequency was found between infertile patients and a control group. It is suggested that in some populations the gr/gr deletion might be an inconsequential polymorphism.

Luteinizing hormone concentrations after gonadotropin-releasing hormone antagonist administration do not influence pregnancy rates in in vitro fertilization-embryo transfer.

OBJECTIVE: To determine the impact of circulating LH concentrations during controlled ovarian hyperstimulation on the outcome of IVF. DESIGN: Retrospective study. SETTING: University hospital. PATIENT(S): Two-hundred seventy women who had a short stimulation protocol with GnRH antagonist and ovarian stimulation with recombinant FSH (rFSH). INTERVENTION(S): GnRH antagonist and rFSH were administered SC; blood samples were collected on the day of GnRH antagonist administration, 1 day after, and on the day of hCG administration. MAIN OUTCOME MEASURE(S): A threshold of 0.5 IU/L on the day of hCG was chosen to discriminate between women with LH concentrations 0.5 IU/L (group B, n = 151). RESULT(S): The two groups were comparable with regard to the clinical parameters. In group A, significantly lower LH concentrations were observed on day 9 of the cycle and on the day of hCG administration. The numbers of oocytes retrieved, embryos obtained, and embryos cryopreserved were significantly higher in group A compared with group B. The proportion of clinical pregnancies was similar in the two groups (21.1% vs. 22.7 % per ET). CONCLUSION(S): In GnRH antagonist and rFSH protocols, suppressed serum LH concentrations do not have any influence on the final stages of follicular maturation, pregnancy rates, or outcomes.