Effects of lead, cadmium, copper and zinc levels on the male reproductive function.

This study aimed to investigate the effects of heavy metals on measures of male fertility. One hundred and two infertile men with occupational exposure and thirty fertile men were included in this study. Blood and urinary levels of lead, cadmium, zinc and copper were measured by atomic absorption spectrophotometry. Semen parameters and a motile sperm organelle morphology examination were also performed. Measures of hormonal levels, oxidation-reduction potential, DNA fragmentation index and chromatin condensation were assessed for all participants. Heavy metals levels, oxidative stress and DNA quality were significantly higher in the infertile group compared to controls. FSH and testosterone levels were lower in the infertile group. A urinary cadmium level was positively associated with abnormal sperm morphology (r = .225, p < .05). Normal morphology was inversely correlated with the duration of the exposure (r = -.227, p = .022). The blood lead level was positively related to the level of testosterone (r = .223, p = .031). Cadmium and lead blood levels were positively correlated with the level of chromatin decondensation (r = .528, p < .001; r = .280, p = .017). Our study showed that occupational exposure to heavy metals is very harmful to reproductive health. DNA quality and oxidative stress investigations must be recommended for reprotoxic exposed patients prior to in vitro fertilisation treatment.

Y chromosome microdeletions screening in Tunisian infertile men.

The aim of this study was to establish the prevalence of chromosomal abnormalities and microdeletions on the Y chromosome in Tunisian infertile men with severe oligozoospermia or non-obstructive azoospermia. In cases of azoospermia, we aimed also to correlate histological results after negative testicular sperm extraction with the type of Y chromosome microdeletion. 84 infertile patients and 52 controls were screened for karyotypic abnormalities using G-banding and Yq chromosome microdeletions using multiplex PCR. 7 infertile males (8.3%) carried chromosomal abnormalities and 8 (9.5%) presented Y chromosome microdeletions. The frequency of chromosome abnormalities in azoospermic patients was 11.1% vs 3.3% in the severe oligozoospermic group. Klinefelter syndrome was the most frequent chromosomal abnormalities in 85.7% of cases. Only one patient had a 46,X,del Y/45,X karyotype. The frequency of microdeletions was 11.1% in the azoospermic group and 6.7% in the severe oligozoospermic group. Six out of 84 (7.14%) of the infertile patients had microdeletions in the AZFc region, one azoospermic male had microdeletion in the AZFbc regions and one in the AZFb region, no deletions in the AZFa region. Among the 6 azoospermic patients with microdeletions: 4 had Sertoly cell only syndrome (SCOS) and 2 had maturation arrest (MA). Genetic abnormalities in infertile Tunisian patients are similar to those reported in other countries. The knowledge of the existence of genetic abnormalities and microdeletions is useful to provide a correct diagnosis and it allows the clinician to refer the patient to adequate assisted reproduction technique and examine the value of testicular biopsy pertinence.

Basal follicle stimulating hormone level correlated to age is a good prognostic criterion for the outcome of intracytoplasmic sperm microinjection.

BACKGROUND: Age and increased FSH serum level in women are prognosis criteriae associated with decreased fertility. OBJECTIVE: The aim of this study was to investigate whether age-specific FSH concentration can be a predictor of the outcome of ovarian stimulation in women undergoing IVF. METHODS: A total of 676 women undergoing their first IVF cycle over a 3-year period were included in this retrospective cohort study. Patients were grouped according to age (< or ≥  38 years), and within each age range, patients were grouped into bFSH quartiles (< or ≥  9.6 mUI/L). We have considered four study groups: group A (Age < 38 years and FSH < 9.6 m UI/l), group B (Age < 38 years and FSH ≥ 9.6 m UI/l), group C (Age ≥ 38 years and FSH < 9.6 m UI/l), group D (Age ≥ 38 years and FSH ≥ 9.6 m UI/l). The outcome measures in each group included: consumed quantity of gonadotrophin, poor response, cycle cancellation, oocyte yield, number of embryos obtained, embryonic quality (grade 1 embryo), as well as, fertilization, implantation, clinical pregnancy and childbirth rates. Analysis of the Results compares laboratory parameters and ICSI Results, based on a statistical analysis that is essentially descriptive. RESULTS: High bFSH levels in young patients (< 38 years) predicts a higher poor response (p < 0.0001), higher stopped cycles (p < 0.0001), lower oocyte yield (p < 0.0001) and lower embryos obtained (p < 0.0001) in IVF cycles but does not translate to either lower pregnancy, childbirth or implantation rates. In old women high FSH level does not influence ICSI outcome but may increase poor response (p <0.01) and stopped cycles (p < 0.0001). In each age group, the rate of spontaneous miscarriage does not increase according to FSH level. The pregnancy rate and child birth rate are better in young women with high FSH levels than in older women with normal FSH levels (p < 0.05). CONCLUSION: The findings of this study suggest that basal FSH concentrations when correlated to age is a good predictive factor of ovarian response for assisted reproductive treatment. In young women a high FSH level may affect laboratary parametres but not pregnancy rate. In old women normal FSH level does not improve ICSI outcome but may increase "avorted" cycles. Pregnancy rate and child birth rate are better in young women with high FSH levels than in older women with normal FSH levels.

Progesterone for Luteal Phase Support in In Vitro Fertilization: Comparison of Vaginal and Rectal Pessaries to Vaginal Capsules: A Randomized Controlled Study.

BACKGROUND: In IVF, Luteal phase support is usually performed using vaginal progesterone. A part of patients using this route reports being uncomfortable with this route. We tried to study whether the rectal route could be an effective alternative and associated with less discomfort. PATIENTS AND METHODS: A prospective randomized controlled study. All patient were eligible for IVF treatment for infertility. After oocyte pickup, 186 patients were allocated to one the following protocols for luteal phase support: (i) rectal pessaries group: natural progesterone pessaries administered rectally 200 mg three times a day, (ii) vaginal pessaries group: natural progesterone pessaries administered vaginally 200 mg three times a day), and (iii) vaginal capsules group: natural micronized progesterone capsules administered vaginally 200 mg three times a day. On the day of pregnancy test, patients were asked to fill in a questionnaire conducted by an investigator in order to assess the tolerability and side effects of the LPS treatment taken. The primary endpoint was the occurrence of perineal irritation. RESULTS: Fifty eight patients were assigned to the rectal pessaries group, 68 patients to the vaginal pessaries group, and 60 patients to the vaginal capsules group. All patients adhered to their allocated treatment. Implantation and clinical pregnancy rates per transfer did not differ between the three groups. Perineal irritation, which was our primary endpoint, was the same for all the three groups (respectively 1.7 % versus 5.9 % versus 11.7%). Regarding the other side effects, more patients experienced constipation and flatulence with the rectal route, whereas more patients reported vaginal discharge in the vaginal capsules group. CONCLUSION: Rectal administration for luteal phase support is effective and well accepted alternative to vaginal route.

Failure of in vitro fertilization: prognosis criteriae.

BACKGROUND: age, obesity and increased FSH serum level in women are prognosis criteriae associated with decreased fertility and adverse Assisted Reproductive Technologies (ART) outcomes. OBJECTIVE: To assess the effect of age, FSH and BMI on pregnancy rate in ICSI. METHODS: A retrospective and comparative study of 500 women who underwent ICSI cycle during the study period from January 2004 to December 2005. Age, FSH and BMI were compared in two groups of patients: Those achieving a pregnancy: The "pregnancy+" group and those failing to have a pregnancy: The "pregnancy-"group. For each of previous parameters ROC curve and logistic regression study were performed. RESULTS: age was significantly lower in "pregnancy+" group (32,4±3,9 years vs 33,7±4,8 ans ; p=0,005). Analysis of ROC curve and logistic regression study show that for age, the most discriminative cut-off for predicting pregnancy is 38 years (Se=7,5%, Sp=75,6%) (AUC=0,572; p=0,02) (OR=2,1 ; LR+=6,7 ;IC[1 ;1,4] ; p0,009). FSH was significantly lower in "pregnancy+" group (5,5±1,8UI /L vs 6,2±3UI/L, p=0,003). Analysis of ROC curve and logistic regression study show that for FSH, the most discriminative cut-off for predicting pregnancy is 9UI/L (Se=3,7%, Sp=85.2%) (AUC=0,539 ; p=0,03) (OR=3,6; IC[1,4 ;9,3]; LR+ =10,1; p=0,003). BMI was also significantly lower in "pregnancy+" group (24,7± 3,6 kgm-2 vs 27,1±4,5 kgm-2 ; p<10-3). The most discriminative cut-off for predicting pregnancy is 25,4 kgm- 2 (Se=31,7%, Sp=33,3%) (AUC=0,663 ; p<10-3) (OR=4; IC[2,1 ;7,7]; LR+ =19,38; p<10-3). CONCLUSION: age, FSH and BMI affect markedly the prognosis of ICSI. We found significantly lower Pregnancy rates in older women (> 38 years), in women with elevated FSH (> 9UI/L) or elevated BMI (> 25,4 kgm-2). Our results can be used when counseling and before including patients in an IVF program, to give them probability of success and weight loss required to optimize chances of pregnancy.

Impact of gamete cell quality on the outcome of intracytoplasmic sperm injection.

BACKGROUND: the quality of the gametes used for an intracytoplasmic microinjection of spermatozoïde is a significant factor which can influence pregnancy rates. AIM: To assess the effect of conventional sperm parameters, origin of spermatozoa and oocyte quality on pregnancy rate in ICSI. METHODS: A retrospective and comparative study of 500 women who underwent ICSI cycle during the study period from January 2004 to December 2005. Conventional sperm parameters (count, motility and morphology) and oocyte quality (mature and immatures oocytes) was compared in two groups of patients: Those achieving a pregnancy: The "pregnancy+" group and those failing to have a pregnancy: The "pregnancy-" group. RESULTS: Among the conventional sperm parameters, only spermatozoa count after preparation was significantly higher in "pregnancy+" group (p=0,02). We found significantly more pregnancies in ejaculated and epididymal sperm groups than in the testicular one (p<10-3). The number of oocyte retrieved was significantly higher in "pregnancy+" group (13,9±7 vs 10,6± 7,6 ; p<10-3) with mainly mature oocyte (metaphaseII) (9,1±5,5 vs 6,6±5,4 ; p<10-3). Analysis of ROC curve and logistic regression study show that for mature oocyte, the most discriminative cut-off for predicting pregnancy is 4 (OR=2,1 ; LR+=6,7 ;IC[1 ;1,4] ; p0,009). CONCLUSION: Conventional parameters of ejaculated sperm have almost no influence on pregnancy rates in ICSI. Testicular sperm seem to have worse results. The number of oocytes retrieved and the proportion of mature oocytes (metaphaseII) affect markedly the prognosis.

Identification of a new recurrent aurora kinase C mutation in both European and African men with macrozoospermia.

STUDY QUESTION: Can we identify new sequence variants in the aurora kinase C gene (AURKC) of patients with macrozoospermia and establish a genotype-phenotype correlation? SUMMARY ANSWER: We identified a new non-sense mutation, p.Y248*, that represents 13% of all mutant alleles. There was no difference in the phenotype of individuals carrying this new mutation versus the initially described and main mutation c.144delC. WHAT IS KNOWN ALREADY: The absence of a functional AURKC gene causes primary infertility in men by blocking the first meiotic division and leading to the production of tetraploid large-headed spermatozoa. We previously demonstrated that most affected men were of North African origin and carried a homozygous truncating mutation (c.144delC). STUDY DESIGN, SIZE, DURATION: This is a retrospective study carried out on patients consulting for infertility and described as having >5% large-headed spermatozoa. A total of 87 patients are presented here, 43 patients were published previously and 44 are new patients recruited between January 2008 and December 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients consulted for primary infertility in fertility clinics in France (n = 44), Tunisia (n = 30), Morocco (n = 9) or Algeria (n = 4). Sperm analysis was carried out in the recruiting fertility clinics and all molecular analyses were performed at Grenoble teaching hospital. DNA was extracted from blood or saliva and the seven AURKC exons were sequenced. RT-PCR was carried out on transcripts extracted from leukocytes from one patient homozygous for p.Y248*. Microsatellite analysis was performed on all p.Y248* patients to evaluate the age of this new mutation. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a new non-sense mutation, p.Y248*, in 10 unrelated individuals of European (n = 4) and North African origin (n = 6). We show that this new variant represents 13% of all mutant alleles and that the initially described c.144delC variant accounts for almost all of the remaining mutated alleles (85.5%). No mutated transcripts could be detected by RT-PCR suggesting a specific degradation of the mutant transcripts by non-sense mediated mRNA decay. A rare variant located in the 3' untranslated region was found to strictly co-segregate with p.Y248*, demonstrating a founding effect. Microsatellite analysis confirmed this linkage and allowed us to estimate a mutational age of between 925 and 1325 years, predating the c.144delC variant predicted by the same method to have arisen 250-650 years ago. Patients with no identified AURKC mutation (n = 15) have significantly improved parameters in terms of vitality and concentration of normal spermatozoa, and a decreased rate of spermatozoa with a large head and multiple flagella (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Despite adherence to the World Health Organization guidelines, large variations in most characteristic sperm parameters were observed, even for patients with the same homozygous mutation. We believe that is mainly related to inter-laboratory variability in sperm parameter scoring. This prevented us from establishing clear-cut values to indicate a need for molecular analysis of patients with macrozoospermia. WIDER IMPLICATIONS OF THE FINDINGS: This study confirms yet again the importance of AURKC mutations in the aetiology of macrozoospermia. Although a large majority of patients are of North African origin, we have now identified European patients carrying a new non-sense mutation indicating that a diagnosis of absence of a functional AURKC gene should not be ruled out for non-Magrebian individuals. Indirect evidence indicates that AURKC might be playing a role in the meiotic spindle assembly checkpoint (SAC) during meiosis. We postulate that heterozygous men might have a more relaxed SAC leading to a more abundant sperm production and a reproductive advantage. This could be the reason for the rapid accumulation of the two AURKC mutations we observe in North African individuals. STUDY FUNDING/COMPETING INTEREST(S): None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the programme GENOPAT 2009 from the French Research Agency (ANR).

[Basal fsh level is only predictive of the quantitative aspect of the ovarian response]. / Le taux basal de FSH à j3 du cycle est un facteur prédictif de l'aspect quantitatif de la réponse ovarienne à la stimulation.

BACKGROUND: Determination FSH and LH at day 3 of the menstrual cycle predicts the response to stimulation. AIM: To evaluate the value of FSH and LH measurements compared with women's age in predicting qualitative and quantitative ovarian response to gonadotrophin stimulation. METHODS: 305 patients underwent at least one intra cytoplasmic sperm injection (ICSI) cycle. The levels of FSH and LH at day 3 were determined in an earlier cycle. A good quantitative ovarian response was defined as ³3 oocytes retrieved and 3 embryos obtained. A good qualitative ovarian response was defined as a percentage of mature oocytes ³75% and immature ones²15% of the total number of oocytes retrieved with at least one top quality embryo obtained. RESULTS: Receiver operating characteristic (ROC) curves were generated for FSH, LH and female age. FSH is better than female age in predicting the number of oocytes retrieved (respectively ROCAUC=0.77, p=10-3 versus ROCAUC=0.73, p=10-3) and the number of embryos obtained (ROCAUC=0.69, p=10-3 versus ROCAUC=0.66, p=10-3). LH is non predictive. None of the three tested parameters was predictive of the fertilization and pregnancy rates. An FSH cutoff was calculated and a value of 7.8mUI/ml is associated with a sensitivity of 73% and a specificity of 70% for the prediction of ovarian response to controlled stimulation. CONCLUSION: Basal FSH level predicts good quantitative rather than qualitative response. LH is non predictive. FSH and LH do not predict pregnancy rate. Patients having high FSH levels should not be excluded from IVF/ICSI treatment.

MLPA and sequence analysis of DPY19L2 reveals point mutations causing globozoospermia.

STUDY QUESTION: Do DPY19L2 heterozygous deletions and point mutations account for some cases of globozoospermia? SUMMARY ANSWER: Two DPY19L2 heterozygous deletions and three point mutations were identified, thus further confirming that genetic alterations of the DPY19L2 gene are the main cause of globozoospermia and indicating that DPY19L2 molecular diagnostics should not be stopped in the absence of a homozygous gene deletion. WHAT IS KNOWN ALREADY: Globozoospermia is a rare phenotype of primary male infertility characterized by the production of a majority of round-headed spermatozoa without acrosome. We demonstrated previously that most cases in man were caused by a recurrent homozygous deletion of the totality of the DPY19L2 gene, preventing sperm head elongation and acrosome formation. In mammals, DPY19L2 has three paralogs of yet unknown function and one highly homologous pseudogene showing >95% sequence identity with DPY19L2. Specific amplification and sequencing of DPY19L2 have so far been hampered by the presence of this pseudogene which has greatly complicated specific amplification and sequencing. STUDY DESIGN, SIZE, DURATION: In this cohort study, 34 patients presenting with globozoospermia were recruited during routine infertility treatment in infertility centers in France and Tunisia between January 2008 and December 2011. The molecular variants identified in patients were screened in 200 individuals from the general population to exclude frequent non-pathological polymorphisms. PARTICIPANTS/MATERIALS, SETTING, METHODS: We developed a Multiplex Ligation-dependent Probe Amplification test to detect the presence of heterozygous deletions and identified the conditions to specifically amplify and sequence the 22 exons and intronic boundaries of the DPY19L2 gene. The pathogenicity of the identified mutations and their action on the protein were evaluated in silico. MAIN RESULTS AND THE ROLE OF CHANCE: There were 23 patients who were homozygous for the DPY19L2 deletion (67.6%). Only eight of the eleven non-homozygously deleted patients could be sequenced due to poor DNA quality of three patients. Two patients were compound heterozygous carrying one DPY19L2 deleted allele associated respectively with a nonsense (p.Q342*) and a missense mutation (p.R290H). One patient was homozygous for p.M358K, another missense mutation affecting a highly conserved amino acid. Due to the localization of this mutation and the physicochemical properties of the substituted amino acids, we believe that this variant is likely to disrupt one of the protein transmembrane domains and destabilize the protein. Overall, 84% of the fully analysed patients (n = 31) had a molecular alteration of DPY19L2. There was no clear phenotypic difference between the homozygous deleted individual, patients carrying a point mutation and undiagnosed patients. LIMITATIONS, REASONS FOR CAUTION: Globally poor fertilization rates are observed after intracytoplasmic sperm injection of round spermatozoa. Further work is needed to assess whether DPY19L2 mutated patients present a better or worse prognostic than the non-diagnosed patients. Evaluation of the potential benefit of treatment with a calcium ionophore, described to improve fertilization, should be evaluated in these two groups. WIDER IMPLICATIONS OF THE FINDINGS: In previous work, deletions of DPY19L2 had only been identified in North African patients. Here we have identified DPY19L2 deletions and point mutations in European patients, indicating that globozoospemia caused by a molecular defect of DPY19L2 can be expected in individuals from any ethnic background. STUDY FUNDING/COMPETING INTEREST(S): None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the program GENOPAT 2009 from the French Research Agency (ANR).

Examination of viability and quality of ovarian tissue after cryopreservation using simple laboratory methods in ewe.

BACKGROUND: The objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezing-thawing procedures. METHODS: Ewe's ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups: fresh tissue (control group) and frozen tissue.In the first part of the study, the follicles viability was assessed by trypan blue staining, calcein AM/ethidium homodimer-1 staining (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes) and morphology in the two groups. In the second part of the study the quality of the whole ovarian tissue was evaluated by the quantification of the release of lactate dehydrogenase measurement (Cytotoxicity Detection kit ROCHE), DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) in primordial and primary follicles (ApopDETEK Kit system Enzo) and morphology in the two groups. 100 Follicles (primordial and primary) were counted on both fresh and frozen hemiovary to assess this various tests. RESULTS: Ovarian follicle viability assessment was similar using trypan blue or calcein/ethidium staining. Follicles showed a decreased viability after freezing-thawing.After cryopreservation, a significant correlation between the percentage of normal follicles and viability rate was found using trypan blue (r=0.82, p<0.05) or calcein AM/ethidium homodimer-1 staining (r=0.76, p<0.05). Increased cytotoxicity showed by enhancement of LDH release was found after cryopreservation (21.60+/-1.1% vs 52.2+/-7.7%). A significant negative correlation between the percentage of morphologically normal follicles and cytotoxicity was observed. No significant difference in DNA fragmentation rate between frozen and control groups was found (26±8.2% vs 38±4.5%). CONCLUSION: We suggest the use of trypan blue staining for the histological assessment of viability, the use of LDH assay for the cytotoxicity assessement and finally the use of DNA fragmentation assessment to valid different freezing-thawing protocols.

A recurrent deletion of DPY19L2 causes infertility in man by blocking sperm head elongation and acrosome formation.

An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization.

[A retrospective study of 339 ICSI cycles: assesment of the two first years of activity of the Assisted Reproductive Technology Center of Aziza Othmana Hospital]. / Etude retrospective de 339 cycles d'injection intra cytoplasmique de spermatozoïdes: bilan des deux premières années d'activité de l'unité d'assistance médicale à la procréation de l'hôpital Aziza Othmana.

BACKGROUND: Intracytoplasmic sperm injection (ICSI) is a micromanipulation-assisted fertilization, whereby one spermatozoon is injected into the oocyte cytoplasm. Initially, ICSI was the treatment of choice for male factor infertility. However, because of the high fertilization and pregnancy rates achieved with this technique, the scope of the procedure has been widened to include couples with other causes of infertility. AIM: The aim of this study was to study the progression of the activity of the assisted reproductive technology's center of Aziza Othmana's Hospital and the ICSI results during the first two years. METHODS: Our study included 269 infertile couples who underwent 339 ICSI cycles between 1st May 2001 and 30 April 2003. Cycles with no oocytes obtained at the follicular aspiration and women aged over 40 years were excluded from this study. RESULTS: The number of ICSI cycles progressed in our center: 150 ICSI cycles in the 1st year, 189 ICSI cycles in the 2nd year. The mean number of picked-up oocytes was 8,8 +/- 5,6. The fertilization rate was 62%. The mean number of transferred embryos was 3,1 +/- 1,5. The pregnancy rate per transfer was 32,4%. The miscarriage rate was 28,4%. The take home baby rate was 67,9%. CONCLUSION: The number of couples undergoing ICSI cycles in our center is increasing. The fertilization rates and pregnancy rates in our center are similar to those published in the literature.