[Association between sperm abnormalities and occupational environment among male consulting for couple infertility]. / Association entre anomalies spermatiques et environnement professionnel chez les hommes consultant pour infertilité de couple.

Alteration of sperm parameters related to occupational exposures is the subject of several studies, often on a case-control approach. The study populations usually comprise men consulting in infertility clinics for couple infertility. The objective of this review is to identify, from these case-control studies, the main occupational factors that may be associated with altered sperm parameters. We selected 13 articles in the PubMed database. Participation in these studies varied from 61 to 2619 subjects, with great methodological heterogeneity, particularly in the characterization of exposure. The main occupations that appear significantly associated with a risk of altered sperm parameters are workmen, painters, farmers, welders, plumbers and technicians. When analysis focuses on occupational exposures, a significant result is reported for solvents, heavy metals, heat, vibrations and non-ionizing radiation. None of the selected studies has found a link with exposure to pesticides.

[Elective single embryo transfer policy at 48/72 h: which results after fresh transfers and frozen/thawed transfers?]. / Politique de transfert électif d'un embryon à 48/72h: quels résultats après transferts frais et transferts d'embryons congelés ?

OBJECTIVES: To evaluate our elective single embryo transfer policy performed at 48/72h and define predictive factors of pregnancy after frozen/thawed embryo transfer. METHODS: Analysis of 289 elective single embryo transfers (eSET) performed in a selected population in the ART center of Marseille University Hospital from January 2005 to December 2011, as well as the 325 following frozen/thawed embryo transfers performed in this population between May 2005 and December 2012. RESULTS: Cumulative pregnancy rate/oocyte retrieval was of 62.6%; 45% of the couples obtained the birth of at least one child. During this studied period, cumulative pregnancy and delivery rates in the whole population remained stable while multiple delivery rate/delivery clearly decreased. Elective single embryo after frozen/thawed transfer gave satisfying results (24.6% pregnancy/transfer) only in the lack of lysis or in case of mild lysis (1-25%) of the transferred embryo. CONCLUSION: The implementation of an eSET policy gives satisfying results, depending largely on embryo quality. By proposing eSET to a well-targeted population, chosen both on clinical and biological criteria, a clear reduction of cumulative multiple delivery rate/delivery was obtained in our center over this period, without any global decrease of cumulative pregnancy rate/attempt. Embryo quality is a major factor of success, especially in frozen/thawed cycles. The elective single embryo frozen/thawed transfer should be carried out only if embryo lysis after thawing does not exceed 25%.

Sperm cryopreservation before cancer treatment: a 15-year monocentric experience.

Sperm banking is an important procedure to preserve fertility before cancer therapy. The aim of this study was to comprehensively analyse cryopreservation activity retrospectively for 1080 patients referred to the sperm bank for sperm cryopreservation before cancer treatment. This study included 1007 patients diagnosed with testicular cancer (TC) (41.7%), lymphoma (26%), other haematological cancers (9.4%) or other types of cancer (22.8%); of these, 29 patients did not produce any semen sample and cryopreservation was impossible for 67 patients. Semen characteristics before treatment were within normal ranges, except moderate asthenospermia. Sperm concentration was significantly lower in TC than in non-TC. Straws from 57 patients (6.3%) were used in assisted reproductive technologies, which led to a 46.8% cumulative birth rate. Straws were destroyed for 170 patients (18.7%) and 140 patients performed semen analyses after cancer therapy. After an average delay of 22.5 months after the end of therapy, 43 patients (30.7%) exhibited azoospermia. This study of a large population of cancer patients revealed a high level of successful sperm storage. Utilization of cryopreserved spermatozoa led to good chances of fatherhood. Nevertheless, sperm banks should be aware of the low rates of straw use and straw destruction by cancer patients.

[Do couples who procreate through sperm donation inform their children?]. / Les couples qui procréent par don de sperme en informent-ils leurs enfants ?

OBJECTIVES: An amendment to the French bioethics law allowing children conceived by gamete donation to know the identity of donors is proposed, while no study can assess the proportion of parents in France that disclose the nature of conception to their donor conceived offspring. The aim of our study was to know whether couples who wish to inform their offspring actually did it. PATIENTS AND METHODS: We sent a questionnaire to parents who had expressed an intention to disclose the nature of conception to their future offspring conceived by sperm donation. This allowed us to evaluate the number of couples who inform their offspring, and the couple and offspring feelings when information was given. RESULTS: Among 38 questionnaires sent, 20 couples answered. Fourteen informed their offspring about the nature of conception, most having lived serenely this moment. 47% of offspring have reacted with indifference. While 19 couples informed their friends or family, six couples did not inform their offspring, and two of them no longer want to disclose anymore. CONCLUSION: Careful thought before the beginning of assisted reproductive technology and support after birth are needed to help couples communicate information to their offspring. Without this communication, any policy of openness to know donor related data seems vain.

[Initial results of a novel technique for sperm retrieval in male infertility due to refractory retrograde ejaculation]. / Résultats préliminaires d'une nouvelle technique de recueil du sperme vésical en présence d'éjaculation rétrograde réfractaire au traitement médical.

AIMS: To describe and evaluate a novel technique of spermatozoa retrieval from patients suffering from infertility secondary to refractory retrograde ejaculation. METHOD: Prospective study to compare mobility and vitality of spermatozoa obtained from urine (U) after oral modification of chemical parameter (PH, Osmolarity) versus from endovesical instillation of sterile spermatozoa culture medium before ejaculation (F). Patients were their own controls. Twelve month prospective follow-up was achieved to document the results of assisted procreation. RESULTS: Eight patients were included and mobility and vitality were improved in all patients after F technique was compared to U technique. With U technique, eight patients on eight had negative defrost test; after F technique, seven patients on eight had a positive defrost test and could therefore have access to assisted reproduction techniques. Four couples had five ICSI and obtained three pregnancies leading to five births. CONCLUSION: Endovesical instillation of sterile spermatozoa culture medium before ejaculation was a safe and effective technique to improve spermatozoa quality in male infertility related to refractory retrograde ejaculation.

[The spinal cord injured patient: semen quality and management by Assisted Reproductive Technology]. / Le blessé médullaire: quel sperme et quelle prise en charge en Assistance médicale à la procréation?

Men with spinal cord injury present a unique infertile population. Only 10 % of them can father children without medical assistance, owing to potential impairments in erection, ejaculation and semen quality. The algorithm typically followed is to retrieve semen by Penile Vibratory Stimulation, in case of failure by Electro Ejaculation. Most of these patients have normal sperm concentrations but abnormally low sperm motility and vitality in the ejaculate. The reasons for poor semen quality in spinal cord injured men are reviewed. If semen cannot be obtained by Electro Ejaculation, or if the ejaculate from Penile Vibratory Stimulation or Electro Ejaculation contains an insufficient quantity or quality of sperm for in vitro fertilization with intracytoplasmic sperm injection, then retrieval of sperm from reproductive tissues is attempted. Despite abnormal semen quality, successful pregnancies with sperm from spinal cord injured male partners have occurred by intravaginal insemination, intrauterine insemination, and in vitro fertilization with intracytoplasmic sperm injection. The prevailing pregnancy and fecundity rates in couples with a spinal cord injured male partner are reviewed.

Ultrasound monitoring in patients undergoing in-vitro fertilization after methotrexate treatment for ectopic pregnancy.

OBJECTIVES: To compare sonographic characteristics of the endometrium and follicles during in-vitro fertilization (IVF) before and after methotrexate (MTX) treatment for ectopic pregnancy. METHODS: This retrospective study, conducted at Conception Hospital from January 2000 to July 2007, included all patients diagnosed with an ectopic pregnancy resulting from IVF treatment that was treated with MTX and who then underwent another IVF cycle. We compared the number and size of follicles and the endometrial thickness and quality on the day of human chorionic gonadotropin injection in the cycles before and after the MTX treatment to determine whether MTX had any effect. RESULTS: Eleven patients were included in the study. The median interval between the IVF cycle resulting in ectopic pregnancy and the first IVF cycle after MTX therapy was 180 (range, 150-900) days. There was no statistically significant difference between the before and after MTX treatment groups with respect to number of follicles (14 (3-20) vs. 9 (4-16), P = 0.12), follicle size (16.5 (14.7-21.7) mm vs. 17.8 (14.9-19.8) mm, P = 0.37), endometrial thickness (10.0 (9.5-12.0) mm vs. 10.0 (7.5-14.0) mm, P = 0.31) or endometrial quality (P = 0.32). Four women became pregnant during the IVF cycle following MTX treatment. CONCLUSIONS: Ultrasound monitoring showed no modification of the characteristics of the endometrium or follicles during IVF after MTX treatment for ectopic pregnancy.

[What are at present the real hopes of pregnancy after ovarian cryopreservation?]. / Quels sont actuellement les espoirs réels de grossesse après cryoconservation ovarienne?

Survival rates for cancers that occur in childhood and adolescence have improved over the last decades, and preservation of future fertility in these patients has become a relevant issue. Premature ovarian failure is a consequence of exposing women to chemotherapeutic drugs and ionizing radiation. Ovarian cryopreservation is an alternative to cryopreservation of embryos or oocytes for theses patients. Ovarian cryopreservation aims to reimplant ovarian tissue after complete remission into the pelvic cavity (orthotopique site) or a heterotopic site like the abdominal wall or the forearm. In vitro folliculogenesis, that aims at the maturation of ovarian cortex primordial follicles cryopreserved for a FIV, is still in an experimental research stage. In this review, the objective was to evaluate the real hopes of pregnancy after ovarian cryopreservation. Indeed, many teams offer ovarian cryopreservation at present time, although only two pregnancies have been achieved to date. In both cases, it can be discussed whether the fertilized oocyte originated from the transplant or from the native ovary. Furthermore, the potential for reintroduction of cancerous cells may limit this technique in cancers that are known to have a risk of ovarian dissemination. The hopes engendered by ovarian cryopreservation, but also its limits, must be explained to the patients before an ovarian surgery for cryopreservation.

Evolution of DNA strand-breaks in cultured spermatocytes: the Comet Assay reveals differences in normal and gamma-irradiated germ cells.

In reproductive toxicity assessment, in vitro systems can be used to determine mechanisms of action of toxicants. However, they generally investigate the immediate effects of toxicants, on isolated germ cells or spermatozoa. We report here the usefulness of in vitro cultures of rat spermatocytes and Sertoli cells, in conjunction with the Comet Assay to analyze the evolution of DNA strand-breaks and thus to determine DNA damage in germ cells. We compared cultures of normal and gamma-irradiated germ cells. In non-irradiated spermatocytes, the Comet Assay revealed the presence of DNA strand-breaks, which numbers decreased with the duration of the culture, suggesting the involvement of DNA repair mechanisms related to the meiotic recombination. In irradiated cells, the evolution of DNA strand-breaks was strongly modified. Thus our model is able to detect genotoxic lesions and/or DNA repair impairment in cultured spermatocytes. We propose this model as an in vitro tool for the study of genotoxic injuries on spermatocytes.

[Ovarian cryopreservation: clinical and ethical problems arisen further to study on cases of ovarian cryopreservation]. / Cryoconservation ovarienne: problèmes cliniques et éthiques. A propos d'une série de cas d'autoconservation de tissu ovarien.

OBJECTIVE: An early ovarian failure and sterility, on patients often still young, may result from some of the anticancerous treatments. Ovarian cryopreservation is a research way for fertility restoration on patients who will have gonadotoxic treatment. The aim of our work was to study the cases of ovarian tissue preserved in our Department, enabling us to assess the clinical and ethical problems of this technique. PATIENTS AND METHODS: A retrospective study was conducted from January 2001 onwards up to October 2005, on 14 patients, whose ovarian cortex was frozen our the University Fertility Center (Hôpital de la Conception, Marseille, France). RESULTS: In our study, the patients' average age was 17+/-5.5 and the median was 14 years [13-24]. Eight patients were under 18 (11+/-3-years-old). The cryopreservation indications were especially haematological (N = 9). More than half of the patients (N = 8) had undergone a gonadotoxic treatment before ovarian cryopreservation. DISCUSSION AND CONCLUSION: Ovarian cryopreservation is still a recent technology in reproductive medicine. The future of ovarian tissue after thawing, with its different techniques of autograft, is still not consensual. Experimental research remains essential to improve the freezing protocols and ovarian transplant in human medicine.

[Optimization of artificial inseminations with donor semen: a four-year experience]. / Optimisation des résultats d'inséminations intra-utérines uniques avec sperme de donneur: bilan de quatre ans d'activité.

OBJECTIVE: Our aim was to analyse the results of a donor insemination program using ovarian stimulation, swim-up sperm preparation and intrauterine insemination proposed to women with a maximum age of 39. Incidence of several clinical and biological parameters on success rates was investigated. PATIENTS AND METHODS: Retrospective analysis of the results of 249 cycles performed in 106 couples during a four-year period is reported. RESULTS: Overall pregnancy rate of 28.1% and delivery rate of 22% per cycle were achieved, with a multiple pregnancy rate of 11.4%. Most of the pregnancies (84%) were obtained before the fourth insemination. Among the different parameters studied the total number of motile sperm inseminated was found to be the most important factor for success rate: pregnancy rate per cycle reached 40.4% if more than 1.5 million progressive sperm were inseminated vs. 24.7% if they were less than 1.5 million (P<0.05). DISCUSSION AND CONCLUSION: In precise conditions, outcome of inseminations with donor semen can reach satisfying pregnancy rates, being a valuable help for couples suffering of long-time infertility.

Meiotic anomalies in infertile men with severe spermatogenic defects.

BACKGROUND: This study was aimed at evaluating the rate of pairing failure in pachytene spermatocytes of patients presenting either an obstructive (O) or a non-obstructive (NO) infertility. METHODS: Forty-one patients and 13 controls underwent testicular biopsy. Among the patients, 19 had an O infertility and 22 a NO infertility. Preparations of all patients and controls were Giemsa-stained, and synaptonemal complexes from nine of these patients and one control were immunostained. RESULTS: In all, 2931 pachytene nuclei were analysed. The mean rate of asynapsed nuclei from the NO group (25.4%) was significantly higher than that of the O group (9.8%). There was no significant difference between the O group and the controls (10.6%). Immunocytochemistry showed that the number of pachytene nuclei decreased from the early to late pachytene sub-stage in all patients. Two NO patients, one azoospermic and one oligozoospermic, had a high percentage of asynapsed nuclei (86 and 91.8% respectively); one of these patients also presented a precocious localized separation of sister chromatids. CONCLUSION: high levels of extended asynapsis could arise from a primary meiotic defect which may be responsible for 9% of the NO male infertilities at our centre. The prevalence of early pachytene substages suggests that the pachytene checkpoint is localized at the mid-pachytene stage in humans.

A truncated HCV core protein elicits a potent immune response with a strong participation of cellular immunity components in mice.

The immunogenicity of a truncated HCV core protein (Co.120) was studied in BALB/c and C57BL/6 mice, given three intramuscular injections of antigen, adjuvanted with either aluminum hydroxide or Freund's adjuvant. A rapid antibody response was noted after the first dose, with both strains of mice eventually exhibiting comparable levels of anti-core IgG (titers >1:100000), with a mixed IgG1/IgG2a subclass response. Spleen cells from Co.120-immunized mice gave a significant specific proliferative response. IFN-gamma gene expression was also detected after an ex-vivo specific stimulation of spleen cells in all immunized mice. This response was independent of dose, H-2 genetic background or type of adjuvant. The results indicated that immunization with the Co.120 protein elicits a potent anti-HCV humoral and cellular immune response.

[Pregnancy outcome and the status of the newborn infant after ICSI at La Conception Hospital in Marseille]. / Evolution des grossesses et état des enfants à la naissance après ICSI à l'hôpital de La Conception à Marseille.

OBJECTIVE: To evaluate obstetric outcome of pregnancies and pediatric follow-up of children born after ICSI procedure. MATERIAL AND METHOD: Prospective study from october 1994 to September 1998 in medical assisted procreations center in La Conception hospital in Marseilles. Three hundred forty-two couples undergoing ICSI procedures. INTERVENTIONS: analysis of pregnancy rates, prematurity rates, obstetric outcome and frequency of congenital malformations. RESULTS: One hundred seventy-eight pregnancies have been obtained with 111 children. Multiple pregnancy rate arise 29.7%. The average term at birth in the singleton pregnancies (38.7 weeks) is higher than in the twins (35.7 weeks). The prematurity rate of delivery before 35 weeks of gestation is about 9.6%. Seven of 111 neonates was born with a congenital malformation, no cardiovascular one. One of boys presents a bilateral cryptorchidism with severe bilateral hypotrophy. CONCLUSIONS: Our results are similar with those of others teams. Congenital malformation rates is near rates reported in others studies. However, no bilateral cryptorchidism with bilateral severe hypotrophy has been yet reported in literature.

[Morphological aspect of embryos obtained after fertilization in vitro for male factor or ICSI]. / Aspect morphologique des embryons obtenus après une fécondation in vitro pour indication masculine ou ICSI.

OBJECTIVE: In order to study the eventual impact on fertilization and embryo characteristics of the microinjection procedure we compared the quality of the embryos obtained by ICSI with those of in vitro fertilization with male factors (MF IVF). MATERIAL AND METHODS: One hundred thirty-four cycles of IVF treatment (group 1) were selected with oligoasthenozoospermia according to WHO criteria with a total number of motile spermatozoa between 500,000 and 1 million. One thousand eighty-eight mature oocytes and 486 embryos were obtained. One hundred forty-three cycles of intracytoplasmic sperm injection (group 2) were performed in couples whose in vitro fertilization was imparticable because of extreme sperm impairment. One thousand one hundred forty-seven mature oocytes were injected and 626 embryos were obtained. RESULTS: In group 1, the pregnancy rate per embryo transfer and the implantation rate were respectively 22.7% and 12.3%. In group 2, the pregnancy rate per embryo transfer was 37.1% and the implantation rate was 17%. The statistical analysis of the embryos obtained in the two different groups did not demonstrate any difference in the distribution of the more regular and less fragmented embryos (group A) and those of the more irregular and fragmented embryos (group B). No statistical difference was demonstrated in the chronology of the division of these embryos (groups 1 and 2). CONCLUSION: The pregnancy rate by cycle and by transfer reported by ICSI (p < 0.003 and p < 0.015 respectively) could be related to a significantly higher mean number of transferred embryos (2.65 vs 2.02) in probable relation with a higher cleavage rate (p < 0.00001).

Failure of pregnancy after intracytoplasmic sperm injection with decapitated spermatozoa: case report.

The case of a couple with a history of long standing primary infertility is reported in which the man presented with a decapitated sperm defect. The woman had a normal history and presented with normal clinical characteristics. The couple underwent one unsuccessful conventional in-vitro fertilization (IVF). Subsequently, embryos were obtained and transferred after assisted fertilization attempts: in all, three subzonal inseminations and four intracytoplasmic sperm injections. A total of 49 mature oocytes was injected in both studies, 25 embryos obtained and 20 embryos transferred, three of them after freezing and thawing. Despite the good embryo morphology, implantation was unsuccessful and no pregnancy occurred. The failure of implantation may have resulted from an arrest in early embryonic development related to the sperm anomaly. One hypothesis is that transferred embryos may carry a chromosomal imbalance that prevents them from progressing to the blastocyst stage. Nevertheless, we cannot exclude the possibility that the woman is responsible for the implantation failure. Co-culture associated with a further attempt could provide information regarding the ability of embryos to progress to the blastocyst stage and implant.

Ultrastructural and immunocytochemical evidences of core-particle formation in the methylotrophic Pichia pastoris yeast when expressing HCV structural proteins (core-E1).

Particulate antigens of the Hepatitis C virus (HCV) are reported for the first time by transmission electron microscopy in Pichia pastoris. The yeast was cloned to express the first 339 NH2-terminal amino acids of the HCV polyprotein (C-E1.339 polypeptide). The C-E1.339 polypeptide covers the putative 191 aa of the core protein (aa 1-191) and 148 aa of the E1 envelope antigen (aa 192-339). Virus-like particles (VLP) with diameters ranging from 20 nm to 30 nm were specifically observed in those cells expressing the HCV polyprotein. The VLP appeared along the membrane of the endoplasmic reticulum, but were fundamentally localized in vacuoles, either free or inside autophagic bodies. Clustered particles, chains of particles, high-density reticular structures, and crystalloid bodies were also detected, the last one being an orderly arrangement of particles with 20 nm diameters. The crystal-associated particles are well differentiated from the intracellular VLP because of their uniform size and shape. We argue that membrane components are retained in the architecture of the VLP, conferring to this particle certain heterogeneity. Both kinds of particles, the VLP formed after treatment with NP-40 and the crystal-associated particles, were core protein-positives. Whether they reflect mature HCV nucleocapsid or intermediary states in the viral nucleocapsid morphogenesis remains unknown. We conclude that, like mammalian cell lines, the P. pastoris yeast could be an appropriate host for the analysis of HCV polyprotein processing and, eventually, virus assembly.

[Intrauterine insemination with the husband's sperm: conclusions of five years experience]. / Inséminations intra-utérines avec sperme du conjoint (IIU-AC): conclusions de 5 ans d'expérience.

We analyzed retrospectively 936 intrauterine insemination (IUI) cycles with husband's sperm (384 couples). Superovulation and induction monitoring occurred in the majority of cases; IUI was timed 36-40 h after ovulatory hCG. The overall pregnancy rate per cycle (PR/C) was 11.4% (107/936). Data analysis demonstrated that PR/C decreased with infertility duration, woman's age (especially after 38 years old) and number of attempts (significantly after the 4 th cycle). Superovulation (in particular by antiestrogens) and induction monitoring seemed to provide an increase in cycle pregnancy rate. Highest PR/C were observed in dysovulation and male infertility groups. In cases of sperm defects, our data showed that 2 parameters had a significant influence on pregnancy outcome: the number of motile progressive inseminated spermatozoa (> 300,000) and the spermatozoa survival rate after 24 h (> 50%). As a conclusion, intrauterine insemination can be proposed as a satisfying treatment of infertility, if precise protocol is followed and its indications well-defined.

Invertase secretion in Hansenula polymorpha under the AOX1 promoter from Pichia pastoris.

A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha. We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H. polymorpha yeast. The culture conditions for invertase production using a fed-batch culture were studied. More than 1.5 x 10(3) U/ml of biologically active invertase (1 g/l) were secreted to the cellular periplasmic space. The fermentative process was scaled up to 50 l. Invertase produced from H. polymorpha was glycosylated, but it contained significantly less carbohydrate than protein produced by S. cerevisiae. Using the Western-blot technique, it was observed that invertase secreted from H. polymorpha and invertase secreted from S. cerevisiae showed common antigenic determinants.

Influence of the morphological aspect of embryos obtained by in vitro fertilization on their implantation rate.

In a series of 500 transfers of embryos obtained by in vitro fertilization, we examined the implantation rate of 1356 embryos transferred in utero. The average implantation rate per embryo was 15.1% and remained relatively constant, regardless of the number of transferred embryos per patient. The implantation rate per embryo, in relation to its morphology, was clearly lower when irregular blastomeres and fragments were present in the perivitelline area. Other embryos, regardless of their morphology, had an identical development potential. Analysis of the results of this series demonstrates the difficulty of determining the development potential of all the embryos on the basis of morphological criteria.