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Investigates the possible link between abnormalities of the blastomeres and apoptosis using annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay to detect cells undergoing apoptosis. Use of annexin V to detect phosphatidylserine exposure; Use of TUNEL assay to test the chromosomal breakdown preceding nuclear collapse of apoptotic nuclei.

The aim of this study was to evaluate the incidence of spermatic aneuploidies in men with severe teratozoospermia and to determine an eventual relation between aneuploidies and a specific morphology of spermatozoa. Fluorescence in situ hybridisation (FISH) using a probe cocktail containing the alpha satellite for the centromeric region of chromosome X, Y and 18 was performed on decondensed spermatozoa from fresh ejaculates of thirty patients with severe teratozoospermia (abnormal forms >80%) and 15 fertile men with normal semen profiles. The mean frequency of teratozoospermia in patients was 91 ± 6.99%. There was statistically a significantly increased frequency of 1818, XY, XX and YY disomies in sperm with severe teratozoospermia compared with normal sperm (1.24% versus 0.08%, 1.42% versus 0.31%, 1.13% versus 0.19% and 1.11% versus 0.24%, respectively, P < 0.001 in all comparisons). The rate of total diploidy was significantly increased in patients compared with controls (1.46% versus 0.16%, P < 0.001). There was a correlation between macrocephalic spermatozoa and diploidy (r = 0.37, P < 0.05). Our data add further evidence that patients with severe teratozoospermia have an increased sperm aneuploidy rate and that this is particularly high in macrocephalic spermatozoa; FISH analysis on sperm could help to improve risk assessment and reproductive counselling in these individuals who are frequently candidates for intracytoplasmic sperm injection (ICSI) as a treatment of their infertility, as the use of ICSI has created consequential debate concerning the genetic risk for the offspring., (© 2011 Blackwell Verlag GmbH.)

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.

Apoptosis has been reported in oocytes and human preimplantation embryos both in vitro and in vivo. BCL-2 family proteins are likely to play a pivotal role in controlling oocyte and early embryo degeneration. However, no BCL-2-related survival factors have been identified that would specifically function during oocyte maturation, after fertilization and during early embryogenesis. Here, we performed a comprehensive tissue expression pattern analysis of the BCL-2 family at the mRNA level. While expression of various members was detected in human oocytes and during early primate embryogenesis, our data indicate that BCL2L10 is the predominant maternally loaded Bcl-2 family transcript, revealing an evolutionary conserved expression profile at the egg-to-zygote transition. We provide evidence that BCL2L10 is associated with the microtubule binding protein translationally controlled tumor protein and mitochondria, with a stage-specific redistribution along the pericortical regulatory ooplasm. In dying oocytes, BCL2L10 colocalized with proapoptotic BAX and neutralization of BCL2L10 accelerated oocyte death. We propose BCL2L10 as a novel and prime candidate related to oocyte maturation, fertility, and embryo developmental competence.

Background: The risk of hepatitis C virus (HCV) transmission during assisted reproductive techniques (ARTs) is still disputed and no report concerning its prospective evaluation is available., Methods: The aim of this 4-year follow-up multicentre study that enrolled 86 HCV-serodiscordant couples was to determine whether a sperm-processing method was able to reduce levels of HCV in semen and the risk of HCV transmission to the newborn. All the men were chronically infected by HCV and 10 of them by human immunodeficiency virus. A total of 181 seminal plasmas and 153 sperm fractions were tested for the presence of HCV RNA., Results: HCV RNA tested positive in 20.4% of the seminal samples. All of the 153 final sperm fractions tested negative for HCV. The detection of HCV RNA in semen was significantly correlated with a high viral load in blood (P < 0.05). The presence of HCV RNA in seminal plasma impaired neither semen parameters nor ART issue. From the 58 couples enrolled effectively in an ART programme, 24 pregnancies and 28 newborns were obtained. All of them tested negative for HCV RNA in blood., Conclusion: These results emphasize the safety of the semen-processing method. The negligible risk of transmitting HCV reduces the value of the systematic analysis of HCV RNA in seminal fractions prior to ART. Since use of this analytical procedure involves the freezing of semen, its avoidance would result in an increase in sperm quality and reduce the need to perform intracytoplasmic sperm injection techniques.

Primordial follicles from different mammal species can survive and enter the growth phase in vitro but do not develop beyond the primary stage. The hypothesis was that, in sheep, in vitro follicular growth is arrested because of a lack of secretion of GDF9 and/or BMP15. Cortical slices of 0.3-0.5 mm thickness issued from 5- to 6-month-old lambs were cultured for 15 days. The pieces were fixed on days 0, 2, 4, 7, 10, and 15 of culture. Follicle morphology, RT-PCR exploration of GDF9 and BMP15 mRNA, immunohistochemical location of their proteins and their receptor BMPRIB and BMPRII were assessed at different time of culture. The mean percentage of primordial follicles decreased from 58.6% (day 0) to 13.4% (day 15) (P<0.01), whereas that of primary follicles increased from 3.2% (day 0) to 31.5% on day 4 (P<0.01), then remained stable until day 15 (35.6%). The percentage of atretic follicles increased from 14.7% (day 0) to 27.1% (day 15) (P<0.05). A few secondary follicles were observed on days 4 and 10, representing 1.0%, and 2.1% of the total number of follicles. GDF9 and BMP15 mRNAs were detected from harvesting (day 0) up to day 15 following culture. At the same time, positive immunoreactions for GDF9, BMP15 and for BMPRIB and BMPRII were also found in oocyte cytoplasm. In conclusion, expression of GDF9, BMP15 and their receptors BMPRIB and BMPRII are detected during in vitro culture of ovine cortical slices., ((c) 2006 Wiley-Liss, Inc.)

In human post-natal somatic cells, low global levels of DNA methylation have been associated with the hypomethylation of several repetitive elements, a feature that has been proposed to be a surrogate epigenetic marker. These data, mainly derived from the analysis of cancer cells, suggest a potential association between loss of cell-growth control and altered differentiation with hypomethylation of repetitive sequences. Partial hydatidiform moles (PHMs) can be used as an alternative model for investigating this association in a non-tumorigenic context. This gestational disease is characterized by abnormal overgrowth and differentiation of the placenta and spontaneous abortion. Here, we comprehensively analyse the DNA methylation of these trophoblastic tissues in both PHM and normal placenta at global and sequence-specific levels. Analysis of the global 5-methylcytosine content and immunohistochemistry indicate that PHM and normal placenta have identical global levels of DNA methylation. In contrast, bisulfite genomic sequencing shows that, whereas Alu, NBL2 and satellite 2 repetitive elements are equally methylated, LINE-1 sequences are hypermethylated in PHM tissues ( approximately 2-fold relative to normal placenta). Interestingly, altered demethylation is also found in triploid diandric embryos that originate from dispermic fertilization of an oocyte, a common event responsible for most PHMs. In conclusion, alterations of DNA methylation do not seem to be randomly distributed in PHM, as several repeated elements remain unaltered, whereas LINE-1 sequences are hypermethylated. In addition, our findings suggest that the hypomethylation of repetitive elements in cancer is directly linked to the neoplasic process and not a simple consequence of loss of growth control observed in most of the cancer cells.

The aim of this study was to analyse the frequency of sex-chromosomal aneuploidy in human spermatozoa of severe oligozoospermic men undergoing intracytoplasmic sperm injection (ICSI), to evaluate the impact of these chromosomal anomalies on the results of the ICSI. Fluorescence in situ hybridization (FISH) with direct label fluorescence DNA probes specific for chromosome X, Y and 18 was performed on decondensed spermatozoa from fresh ejaculates of 12 patients with severe oligozoospermia undergoing ICSI. A total of 500 spermatozoa were analysed per donor. The rate of gonosomal aneuploidy was significantly increased in the patients compared with normal donors (3.5% and 0.8% respectively). The sex-chromosomal anomalies due to meiosis I (XY) are less important than the anomalies caused by meiosis II (XX or XY), but the difference was not statistically significant. There was a negative correlation between the rate of aneuploidy and the percentage of spermatozoa with normal morphology (r = -0.71; P < 0.05). The correlation was negative between the percentage of gonosomic aneuploidy and the rate of fertilization (r = -0.7; P < 0.001). Our results suggest an increased rate of gonosomic aneuploidy in the patients with oligozoospermia compared with the normal population. This aneuploidy, although it decreases the rate of fertilization, does not seem to affect the rate of cleavage, nor the embryonic quality.

Background: Several studies have described geographic variations in human fecundability, but this phenomenon has almost exclusively been studied at an international level rather than within a given country. Our aim was to describe geographic variations in fecundability, the monthly probability of pregnancy, between four cities of France., Methods: We conducted a cross-sectional study in four French maternity units from Toulouse, Rennes, Lyons and Paris, among partners of pregnant women. Women were asked about the time to pregnancy (TTP) of their current pregnancy. TTP was analysed with a discrete Cox model allowing to estimate fecundability ratios (FR)., Results: Time to pregnancy was defined for 894 couples. There was no strong evidence of heterogeneity in fecundability between the four compared cities (p=0.05 without adjustment and p=0.25 after adjustment for behavioural and medical factors). The highest fecundability was observed in Rennes and the lowest in Toulouse (fecundability ratio (FR)=1.28, 95% CI: 1.01-1.63). Differences in fecundability were smaller between the other cities., Conclusion: We highlighted a possibly slightly higher fecundability in Rennes compared to Toulouse. Possible explanations for this finding are discussed. We note that the finding is consistent with previous observations indicating a higher sperm concentration among semen donors in Rennes than in Toulouse.

Background: The aim of this study was to evaluate the long-term outcome of autotransplantation of vitrified warmed hemi-ovaries into ewes., Methods: Six hemi-ovaries from six ewes aged 6 to 12 months were vitrified. After dissection of the medulla, the hemi-ovarian cortex was stored at -196 degrees C in liquid nitrogen. Four to six weeks after the first laparotomy, the left ovary was removed and the vitrified-warmed hemi-ovary was sutured., Results: Plasma progesterone concentration increased in a regular manner in all ewes. Three pregnancies occurred, from which four lambs were born. The first delivery of a normal lamb occurred in February 2003. The second delivery of two normal lambs occurred in March 2003 (a 2.5 kg male and a 2.8 kg female). The last lamb had a normal delivery but had a malformation of the left leg and the oesophagus. This lamb died two months after delivery from pneumariae. Histological examination of the grafted vitrified ovaries showed few primordial and antral follicles., Conclusions: These three pregnancies in a ewe model may indicate that ovarian vitrification gives results as good as those from a slow cooling protocol in autograft. It is impossible to establish a link between the vitrification procedure and the malformation of the last lamb, and further studies are needed to evaluate the feasibility of ovarian vitrification.

Objective: To evaluate a cryopreservation technique by vitrification of whole ovaries with their vascular pedicle in sheep, by using two cryoprotectant solutions., Design: Animal study., Setting: Fertility clinic in a university teaching hospital., Animal(s): Five to 6-month-old ewes., Intervention(s): Whole sheep ovaries with their vascular pedicles were collected at the slaughterhouse and prepared for cryoprotectant toxicity tests and freezing procedures., Main Outcome Measure(s): Follicle viability assessment by trypan blue test and histological examination of ovary and vessel structure., Result(s): No statistically significant difference in follicle viability or normal primordial follicle rates were observed between ovaries exposed or nonexposed to cryoprotectant solutions. Nor was any statistically significant difference observed before and after vitrification with the two cryoprotectant solutions. The decrease in the number of primordial follicles was smaller when frozen-thawed ovaries were treated with VS4 solution containing dimethyl sulfoxide, formamide, and propylene glycol. There were fewer nuclear anomalies and general follicular anomalies with the VS4 solution. Pedicle fractures occurred in most ovaries during thawing (11/15)., Conclusion(s): Cryopreservation of whole ovary by vitrification appears a promising technique in reproductive medicine. The best histologic results were obtained with the VS4 cryoprotectant. Further studies are required to overcome vitrified ovarian vessel fracture.

Background: Reports of a secular decrease in semen quality remain controversial, particularly due to the possibility of selection bias. We aimed to describe the potential bias due to self-selection of volunteers in semen studies involving fecund men., Methods: Using data from the French multicentre study REPRHOM, we compared the characteristics of the partners of pregnant women for three levels of participation: completion of a refusal questionnaire (n = 698), agreement to complete the study questionnaires only (n = 676) and agreement to complete the study questionnaires and give a semen sample (n = 331, 13% of the subjects approached)., Results: Poorly educated men refused more often to participate than highly educated men. Semen providers were more likely to have experienced unfavourable pregnancy outcomes (odds ratio 1.68, 95% confidence interval 1.14-2.49) compared with participants completing the questionnaires only. Time to pregnancy was similar for all participants., Conclusions: This study demonstrates the existence of selection bias in semen studies associated with fertility and socio-demographic characteristics of men. The results of semen analysis for this population sample cannot be extrapolated to the whole population from which the volunteers originate. More information is required on who participates, and participation rates should be reported in semen studies to make it possible to interpret the results correctly.

Animal experiments have shown that cryopreservation of the ovarian cortex, containing primordial follicles, could be used to preserve gametes thereby restoring fertility in humans and animals. During the last 100 years, many hundreds of species have been lost, and a third of the breeding animals are threatened with extinction. To preserve genetic diversity, notably for the conservation of endangered species, it is essential to conserve female and male gametes. Today, biotechnologies such as artificial insemination and embryo transfer are used in breeding programs and are well developed. However, even using these advanced techniques, there are problems due to the limited number of individuals used as the source of gametes, so that the risk of inbreeding is high, even in large populations. To preserve genetic diversity, it is necessary to create gene banks of male and female gametes and embryos, using a very large number of individual donors. Cryopreservation of ovarian tissue could present a means for enlarging the gene pool. Cryopreserved ovarian tissue could be used in auto- or xenografts, or for in vitro maturation (IVM) of primordial follicles. In this review, we describe the processes for cryopreservation of ovarian tissue and the various possibilities for using it.

In France, assisted reproductive technology (ART) for hepatitis C virus (HCV)-infected patients is now subject to strict control after the publication of recent guidelines. Infertile serodiscordant couples (HCV-viraemic men and their seronegative female partners) require special care to carried out in designated 'viral risk' laboratories. Twelve sequential semen samples taken from an HCV chronically infected patient were analysed within 22 months. HCV RNA was detected in all the seminal plasma sampled before antiviral treatment with relatively high viral loads, and in two of the corresponding fractions of motile sperm obtained after a gradient selection, suggesting that a contamination risk by HCV through ART cannot be excluded. When the selection of sperm on a discontinuous gradient was followed by an additional swim-up step, HCV RNA was never detected in the motile sperm suspension that was frozen in highly secure straws. IVF was performed using cryopreserved sperm that tested negative for HCV RNA, resulting in a pregnancy. One month after embryo transfer, testing for HCV RNA and antibodies in the woman gave negative results.

To investigate the risk of transmission of hepatitis C virus (HCV) via semen in assisted reproduction techniques, semen samples from 32 men chronically infected with HCV attending a center for assisted procreation were tested for HCV RNA by a reverse transcription-PCR protocol by using a modified version of the Cobas AMPLICOR HCV assay (version 2.0; Roche Diagnostics). The sensitivity of the test was 40 copies/ml. Four of 32 seminal plasma samples (12.5%) were found to be positive for the presence of HCV RNA. The median HCV load in blood was significantly higher in patients who were found to be positive for the presence of HCV RNA in semen than in those who tested negative (P = 0.02). In one man, seven consecutive seminal plasma samples tested positive for HCV RNA, as did two consecutive motile spermatozoon fractions; the corresponding fractions obtained after migration of the spermatozoa remained negative. Despite the absence of the proven infectivity of virus in semen samples that test positive for HCV RNA, these findings highlight the fact that seminal fluid may exhibit prolonged HCV RNA excretion. The usefulness of HCV RNA detection in both seminal plasma and spermatozoon fractions before the start of a program of medically assisted reproduction in couples in whom the male partner is chronically infected with HCV would need to be evaluated prospectively with a larger population of subjects exhibiting HCV RNA in their semen.

Objective: To evaluate DNA fragmentation in the oocyte of primordial and primary follicles and morphology of these follicles after freezing and thawing of ovarian cortex in sheep using two freezing protocols., Design: Fragmentation of DNA was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique., Setting: Fertility clinic in a large university hospital., Animals: Five- to 6-month-old lambs., Intervention(s): Two-millimeter-thick slices of hemi-ovary cortex were prepared., Main Outcome Measure(s): Histological structure and DNA fragmentation., Result(s): In the frozen fragments, the percentage of morphologically normal follicles was significantly lower for both protocols compared with the case of the control group of fresh fragments. There was no significant difference between the two types of freezing protocols (60.4% +/- 13.2% vs. 68.4% +/- 13.7%). However, the distribution of abnormalities (nucleus, cytoplasm, and nucleus and cytoplasm) was dissimilar. The results of the TUNEL technique for the three groups showed no significant difference, but the percentage of the TUNEL-positive follicles was slightly lower for the frozen fragments for both protocols with respect to the control group., Conclusion(s): The freezing and thawing process of the ovarian cortex does not induce fragmentation of the DNA on the oocyte of primary and primordial follicles.

Objective: To evaluate long-term outcome of autotransplantation of cryopreserved hemi-ovaries into ewes., Design: Animal study., Setting: University fertility center, Hospices Civils de Lyon; and Ecole Nationale Vétérinaire de Lyon., Patient(s): Grivette ewes., Intervention(s): Six hemi-ovaries from 6 ewes aged 6 to 12 months were frozen with a slow cooling protocol using 2 M of dimethyl sulfoxide as cryoprotectant. After dissection of the medulla, the hemi-ovarian cortex was stored at -196 degrees C in liquid nitrogen. Freezing procedure was performed with a programmable freezer. Semiautomatic seeding was performed before crystallization. Four to 6 weeks after the first laparotomy, the left ovary was removed and the frozen-thawed hemi-ovary was sutured., Main Outcome Measure(s): Mean plasma concentrations of FSH, LH, and progesterone after autotransplantation of frozen-thawed hemi-ovary. Ultrasonography was done to confirm pregnancy. Blood samples were collected weekly to measure FSH, LH, and progesterone. After the first birth, the autografted ovary was removed for histologic examination., Result(s): Plasma progesterone concentration increased in a regular manner in all ewes except one 4 weeks after the graft. Concentrations of FSH and LH did not reach the menopausal level. Four pregnancies occurred, from which 6 lambs were born. The first delivery of a normal lamb occurred after 135 days of gestation; the lamb died immediately after birth. The second delivery of two normal lambs occurred after 130 days of gestation. A caesarean section was performed on the third pregnant ewe the 110th days of gestation because the ewe had a vaginal prolapsus. The two normal lambs and the ewe died after surgery. The fourth birth of a normal lamb occurred after 132 days of gestation. Histologic examination of the grafted frozen-thawed ovary showed a regressing corpus luteum and few primordial and antral follicles., Conclusion(s): These four pregnancies in a ewe model may indicate that women who undergo preservation of their ovaries before chemotherapy or radiotherapy can have successful pregnancy.

Objective: To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex., Design: Small follicles (<60 microm in diameter) were isolated enzymatically for viability testing. Dead and live follicles were identified by using trypan blue staining, and follicle morphology was examined histologically., Setting: Centre hospitalo-universitaire de Biologie de la Reproduction, Hôpital Edouard Herriot, Lyon, France., Animal(s): Lambs 5 to 6 months of age., Intervention(s): Two-millimeter slices of hemiovarian cortex were prepared for cryoprotectant toxicity tests and freezing procedures., Main Outcome Measure(s): Follicular mortality and histologic structure., Result(s): For freezing procedures, the concentration of cryoprotectant was increased to 2 M on the basis of results of cryoprotectant toxicity tests in fresh tissues. Follicular mortality rates were 4.6% with of 2 M dimethyl sulfoxide (DMSO) and 3.8% with 2 M of propylene glycol (PROH). After freezing with semiautomatic seeding, follicular mortality rates were 8.4% (2 M of DMSO) and 12.4% (2 M of PROH). Tissue morphology was well preserved with 1.5 M of DMSO or PROH. With 1.5 M DMSO, results of the slow cooling protocol (2 degrees C/min) without seeding and the standard very slow cooling protocol (0.3 degrees C/min) were similar., Conclusion(s): Optimal survival of primordial follicles in the sheep was obtained by using a slow cooling protocol with semiautomatic seeding at 2 M of DMSO.

Objectives: Women with cyclic mastalgia seem to be at risk of fibrocystic breast disease and/or breast cancer. We studied the relationships between mastalgia and hormone levels throughout the menstrual cycle., Study Design: Ostensibly healthy women were monitored during a sum of 326 cycles. A case-control study compared personal and hormonal variables of 30 women experiencing cyclic mastalgia with those of 77 women without this symptom., Results: Except sleeping times, no significant differences were found in personal variables. Cyclic mastalgia and symptoms of fluid retention were slightly associated. Menses and the luteal phase were significantly longer in cases than in controls. Gonadotropin but not ovarian hormone levels were also significantly higher in cases throughout the cycle., Conclusion: Cyclic mastalgia is less related to symptoms of fluid retention or to ovarian hormone levels than to regularly high gonadotropin levels, specific inhibitors might thus be used to alleviate the symptom.

The presence of dead cells in the preimplantation mammalian embryo has been well described. Since Kerr et al. (1972), it has become apparent that these cells die by apoptosis, a form of programmed cell death. This review analyses the recent morphological and biochemical evidence that apoptosis play a role in early mammalian embryo development. Normal and apoptotic (i.e. fragmented) embryos express several apoptosis-related genes during mammalian preimplantation embryo development, with severe changes when apoptosis is activated; these findings support a model in which mammalian preimplantation embryo development is regulated by the ratio of pro- and -anti- apoptotic genes. Apoptosis may be a normal feature in human preimplantation development, even in vivo, and may play an active role in the developing embryo through the removal of genetically abnormal cells. Contrary to these beneficial effects, apoptosis may have detrimental effects if either the number of apoptotic cells or ratio of these cells to the normal cells is elevated. According to this value, embryos could either continue to develop or arrest.

Magainins are antimicrobial peptides with known spermicidal activity. Their activity is inhibited by cholesterol present in eukaryotic cell membranes. Pretreatment of spermatozoa with methyl-beta-cyclodextrin, which extracts cholesterol from cell membranes and induces capacitation, sensitizes them to magainin-2-amide as shown by a decrease in human sperm motility determined by computer-assisted sperm analysis and a concomitant decrease in sperm viability, as measured by MitoTracker(R) Red CMXRos labeling. Magainin-2-amide affects mainly the fast progressive spermatozoa inducing them directly into an immotile state, without an increase in motile non-progressive and slow progressive spermatozoa. We conclude that methyl-beta-cyclodextrin highly potentiates the deleterious effect of magainin-2-amide on human spermatozoa. Most probably, this effect can be explained by cholesterol extraction from sperm cell membranes.

In the present study we describe the localization of proteasomes in human spermatozoa by means of immunolabelling with different monoclonal and polyclonal antibodies detected by confocal microscopy. Western blotting confirmed the specificity of the antibodies and has shown that proteasomes are present in spermatozoa and in seminal fluid. In spermatozoa proteasomes are concentrated in the neck region where the centrioles are located. Some labelling was also detected at the periphery of the head, but no proteasomal antigens were detected in either the nucleus or associated with the flagellum. Proteasome inhibitors did not affect the motility of the spermatozoa, acrosome reaction nor zona binding. It is hypothesized that paternal proteasomes enter the oocyte during fertilization in tight association with the centrioles and may serve a special function during further development which can be associated with the function of a hypothetical proteolysis centre.

In the present study we describe the localization of proteasomes in human oocytes, apoptotic preimplantation embryos, and triploid preimplantation embryos by means of immunolabelling with the MCP21 monoclonal antibody detected by confocal microscopy. While in the oocytes proteasomes are scattered throughout the cytoplasm, in the pronuclear zygote they appear to concentrate at the periphery of the cytoplasm and do not enter the pronuclei. During early cleavage stages, proteasome immunolabelling is concentrated in the nuclei, while the examination of triploid blastocysts showed that proteasomes had a similar cellular distribution to somatic cell lines, i.e. in the nuclei but not in the nucleoli or the cytoplasm. It appears that the distribution of proteasomes dramatically changes during human preimplantation embryo development.

Medical assistance for procreation in a couple where one or both parents has hepatitis C viral infection (HCV) raises the issue of the transmission of the infection to the baby and/or of possible contamination of both the technicians and the gametes or embryos from virus-free parents in the laboratory. It becomes essential to assess transmission risk in assisted reproductive techniques in order to define clearly the management of couples according to their viral status. To define the HCV transmissibility risk in assisted reproduction related to the presence of virus in semen from infected infertile men, HCV RNA detection was performed in sera, and semen and sperm fractions obtained after Percoll gradient centrifugation. HCV RNA was detected in 5% (2/39) of the semen samples tested: in the raw semen, in the seminal fluid and in the cell pellet but never after Percoll selection. According to these results, we suggest a strategy for HCV-infected infertile men who need assistance for procreation.

Objective: To evaluate the effects of freezing, thawing, and autograft of a hemi-ovary on steroid secretion, endometrial maturation, and ovarian histology in ewes., Design: Experimental animal study., Setting: Laboratoire de Zootechnie, Ecole Nationale Vétérinaire, Marcy l'Etoile, France., Animal(s): Six lambs aged 6 months to 1 year old., Intervention(s): Hemi-ovaries were prepared and frozen from the right ovary of six lambs and autografted 4 weeks later to the contralateral ovarian hilus. The autografts and the uterus were recovered 1 year later. Blood tests were performed each week to measure P concentration., Main Outcome Measure(s): Number of primordial follicles; levels of plasma P., Result(s): Histologic examination of ovarian slices after freezing showed no destruction of primordial, primary, secondary, or cavitary follicles. The frozen ovarian autograft showed good recovery of the macroscopic and microscopic ovarian structure. After autografting, histologic examination revealed primordial to cavitary follicles. Secretion of P started to rise 4 weeks after the autograft. Histologic analysis of the endometrium showed numerous glands, vessels, and mucous secretion., Conclusion(s): Frozen ovarian autografts achieved P secretion and endometrial maturity.

Purpose: The improved resolution and optical sectioning of a confocal microscope make it an ideal instrument for extracting three-dimensional information, especially from extended biological specimens such as human embryos. The staining of actin together with chromatin allowed us to specify the architecture of the embryo and the appearance of the nucleus., Methods: F-Actin and chromatin distributions were visualized using laser scanning confocal microscopy in "fresh" and "cryopreserved" human preimplantation embryos obtained by in vitro fertilization., Results: The current study revealed a high rate of multinucleation in arrested or poor-quality embryos (89%, in grade IV embryos)., Conclusions: Confocal microscopy revealed high levels of multinucleated blastomeres, suggesting that the probable cause of arrested development in these embryos was due to multinucleation of blastomeres.

Objective: To evaluate the effects of freezing and thawing on the histologic changes in ovarian fragments from sheep and to determine the feasibility of ovarian autografts., Design: Histologic evaluation of follicles that survived after freezing at -196 degrees C for 2 weeks. Histologic evaluation of ovarian fragments 6 months after the autografts., Setting: Laboratoire de Zootechnie, Ecole National Véterinaire, Marcy l'Etoile, France., Animal(s): Six ewes aged 6 months to 1 year., Intervention(s): Cortical fragments were prepared from the right ovary of 6 lambs and were grafted immediately to the contralateral ovarian hilus or were cooled slowly to -196 degrees C in medium containing dimethyl sulfoxide for 2 weeks. The autografts were recovered 6 months later., Result(s): Histologic examination of ovarian slices after freezing showed no destruction of primordial, primary, secondary, or cavitary follicles. The ovarian autograft showed good recovery of the macroscopic and microscopic ovarian structure. After autografting, histologic examination revealed primordial to cavitary follicles., Conclusion(s): Freezing of ovarian fragments is possible without damaging the follicles. Ovarian autografts showed good recovery of ovarian structure.

Objective: To determine the incidence of cytomegalovirus in the ejaculates of infertile men who were seropositive for IgG antibodies to cytomegalovirus., Design: Prospective study., Patient(s): We tested cytomegalovirus infection in the semen of men participating in an IVF-ET program., Main Outcome Measure(s): IgG and IgM antibodies to cytomegalovirus were measured in sera. We used polymerase chain reaction (PCR) and cell culture to look for both cytomegalovirus DNA and infectious virus in the semen of 70 men with cytomegalovirus-specific antibodies detected in sera., Result(s): Of the infertile couples, 13.5% exhibited "mismatching" serology (i.e., detection of IgG antibodies to cytomegalovirus in male serum only and not in female serum) and constituted a potential risk for cytomegalovirus transmission. Cytomegalovrius was identified in the semen of two patients who were positive for IgG antibodies to cytomegalovirus. Cytomegalovirus DNA also was detected in one positive sample after centrifugation through a three-layer Percoll gradient., Conclusion(s): Human cytomegalovirus was present in the semen from a population of infertile men. Rapid detection can be achieved by molecular techniques such as PCR combined with a hybridization assay. Even though cytomegalovirus was infrequently detected in semen, these data must be considered in determining the risk of transmission and developmental anomalies in infected fetuses.

Laser scanning confocal microscopy (LSCM) is a powerful and useful tool in developmental biology. The diverse applications of LSCM in biomedical field has led to advances in the microscopes themselves and the synthesis of novel specific probes for the observation of biological structures and the hypothesis of physiological process. LSCM was used to visualize the cellular actin cortex together with the chromatin in human arrested preimplantation embryos and in unfertilized oocytes obtained by in vitro fertilization. In embryos, we observed a majority of multinucleated blastomeres and some with fragmented nuclei or anucleated. LSCM also showed nuclei displaying the chromatin condensation and fragmentation patterns suggesting the phenomenon of apoptosis. Moreover, in arrested fragmented embryos of poor morphology, unequal sized blastomeres often showed cellular fragments without nuclei, compatible with aspects of apoptotic bodies.

The effects of co-culture of human spermatozoa with human immortalized endometrial cells - epithelial or stromal - on sperm movement characteristics, including hyperactivation, were studied using computer-assisted sperm analysis (CASA). Epithelial and stromal cell types could be separated following 8-10 days of culture of endometrial cells originating from human biopsies. Both cell types were immortalized by the SV 40 large T antigen. Co-incubation of sperm with epithelial and stromal monolayers enhanced the rate of hyperactivation: 24.9% (P <0.05) and 17.8% (P = 0.05) versus 9.5% as control, respectively, whereas the majority of motility parameters remained unchanged. Conditioned media had no effect upon sperm parameters, including hyperactivation. Co-incubation with either monolayer was able to maintain sperm motility over a longer period than incubation in control medium alone. In four patients whose spermatozoa did not exhibit hyperactivation, co-incubation with epithelial cells, but not conditioned medium, allowed normal rates of hyperactivation (range: 6.9-15.6%).

Co-cultures of human embryos, particularly with Vero cells, are used by several French groups, mainly in cases of successive failures of implantation. In most cases co-culture is continued until the blastocyst stage, expanded if possible. A total of 1603 co-cultures have been performed by 11 groups over a 2-year period. Of these, 41.8% of cleaved eggs (day 2) reached the blastocyst stage at day 5 or day 6. The mean pregnancy rate and implantation rate per transfer were 32.9 and 24.8% respectively, which represented a significant improvement compared to the transfer of 2 day old embryos. The rate of multiple pregnancies remained high (29.1%), which implies that there should be transfer of not more than two blastocysts. The rate of anomalies perceived at birth or in utero was not different from the rate observed in the general population, taking account of the maternal age.

Male infertility can be related to defects in motility, capacitation, acrosome reaction, binding and penetration of the zona pellucida. While different in-vitro techniques (such as micromanipulation which is complicated and expensive) are available for the treatment of male infertility, several pharmacological agents have been shown to increase fertilizing capacity under accurate experimental conditions. Gastrin-releasing peptide (GRP, the mammalian homologue of the amphibian skin peptide bombesin) is present in the reproductive tract and expressed by the pregnant ovine endometrium prior to attachment and throughout the pregnancy. A bombesin-like peptide resulting from alternate splicing of the GRP gene in testis has been detected in primates. In this study, we have tested the ability of GRP to enhance human sperm functions such as motility, capacitation, zona binding and acrosome reaction. Analysis of sperm motility was performed with the ATS 20 computer-assisted semen analysis (CASA) system. Zona binding was analysed using intact human unfertilized oocytes and selective labelling of spermatozoa with two fluorochromes. Our results did not show any positive effect of GRP on these parameters under our experimental conditions. However, when GRP at the concentration of 100 nM was added after ionophore treatment, the percentage of reacted cells increased. significantly (P < 0.05) compared with situations where each agent was used alone. This led us to suppose that the role of bombesin in the different stages of fertilization might not exclude other unknown factors.

Unfertilized oocytes from an in-vitro fertilization programme were stored in different saline solutions and then utilized in a zona binding assay (ZBA). The four saline solutions tested were identical with regard to the capacity of the zona to bind spermatozoa provided by healthy donors. Spermatozoa from 150 infertile patients were tested in the ZBA. The number of spermatozoa bound to the zona correlated positively with sperm concentration, the percentage sperm motility and the percentage of morphologically normal spermatozoa. The population was then divided into two groups according to the level of alpha-glucosidase activity, an epididymal marker. The average number of spermatozoa bound to the zona was diminished in the group with low alpha-glucosidase activity, even when considering strictly equivalent classes of sperm concentration, motility and morphology, respectively.

Spontaneous and ionophore-induced ability of spermatozoa to acrosome-react was examined in asthenozoospermic infertile patients and fertile donors. Spermatozoa were washed free of seminal plasma and capacitated in B2 medium for 2 h at 37 degrees C. Subsequently 5, 10, 20 and 30 microM A23187 (final concentrations) were added to equal aliquots of these samples and incubated for an additional 30 min. The acrosome reaction was then determined by the triple stain technique. The percentage of spontaneous reaction (no ionophore) in asthenozoospermic samples was similar to that in fertile samples (4.2 and 3.8, respectively). However, the ionophore-induced reaction rate remained significantly lower in asthenozoospermic samples than in normozoospermic samples.

In order to evaluate the functional state of human spermatozoa separated on discontinuous Percoll gradient, sperm provided by 15 healthy volunteers were recovered from different layers (60, 70, 80, 90, 100%) of the gradient, then submitted to a complete analysis of sperm movement characteristics. This one was performed using a computer assisted system (Cellsoft, Cryo Resources, Ltd., NY, USA). In all fractions sperm motility was highly maintained during 24 h and all movement characteristics, with the exception of ALH, were different from that observed in seminal plasma. Curvilinear velocity (VCL), linearity (LIN), and percentage of hyper-activated sperm (HA) increased in relation to the Percoll concentration. Nevertheless there was no dramatic difference, concerning motion parameters, between 100%, 90%, and 80% Percoll fractions, which can thereby be pooled in case of insufficient sperm recovering in 100% fraction. On the contrary there is some doubt about the fertilizing ability of spermatozoa recovered in 70% and 60% Percoll layers, since VCL, LIN, and especially HA are severely decreased.

Studies of sperm movement characteristics concern mainly sperm swimming between two glass surfaces (as in the Makler chamber). Using automated videomicrography, (CellSoft, Cryo Resources, New York, USA), we have analysed the movements of human sperm swimming on monolayers of different origins: monkey kidney (Vero) cells, bovine oviduct cells, and human endometrial cells. About 10(5) sperm were deposited upon preparations consisting of monocellular layers adhering to a coverglass, and placed in a deep slide-coverglass system. Experiments were first performed at room temperature then at 37 degrees C. At room temperature, motion characteristics on Vero cell layers (six samples) were not different from those measured in either the conditioned or corresponding non-conditioned media, except for the amplitude of lateral head displacement (ALH) which was significantly lower. Comparison of the three different cell monolayers showed no difference between them for the corresponding motion parameters. The data were dramatically different at 37 degrees C: sperm swimming on cell monolayers of genital origin (oviduct or endometrium) exhibited high rates of hyperactivation (HA: 36.7% and 38.6% respectively), which was significantly more than on either Vero cells (10.9%) or in a control medium (12.6%). Moreover, HA rates were significantly higher on genital cell monolayers than in the corresponding conditioned medium. Hyperactivated sperm exhibited lasting 'star-spin' trajectories rather than 'transitional phases'. It is concluded that passage of sperm on either oviduct or endometrial epithelial cell monolayers can induce sperm hyperactivation and improve their fertilizing capacity.

Hyperactivated motility of capacitated sperm was studied before and after contact for 30 min with the Ca2+ ionophore A23187. Sperm from fertile donors and asthenozoospermic infertile patients were incubated in the presence of increasing concentrations of A23187 in the medium. At 5-10 microM, the ionophore induced a significant increase (P less than 0.05) in the percentage of hyperactivation of sperm from asthenozoospermic subjects. Higher concentrations (20 and 30 microM) were needed to enhance hyperactivation of sperm in the fertile population. In the latter, extended contact with the ionophore induced no significant change compared to a 30-min incubation period. Since this type of movement is an essential feature of the fertilizing gamete, a low hyperactivation rate may partly explain fertilization failure in the asthenozoospermic group.

Two methods for preparing semen (Swim-up migration and centrifugation on a two step density Percoll gradient) were tested in 21 cases of asthenozoospermia (motility less than 30%). The parameters of sperm movement were studied through automated videomicrographic analysis. In 9 cases out of 21 swim-up migration failed while centrifugation on a Percoll gradient failed in only 1 case out of 21.

Glucosidase (alpha G) activity was measured in sperm free seminal plasma from 1200 patients consulting for primary infertility, in whom clinical examination of epididymides revealed some abnormalities and histories of genital infections. They constituted the group with epididymal pathology (P) that was compared with a reference group (R) of 246 men without any epididymal pathology. The distribution of alpha G was significantly different between the two groups, even if we considered only the subjects in group P with normal sperm count (PN: 353 men: p less than 10(-6). 15.9% of subjects in group PN exhibited alpha G values as low as vasectomized men, versus 1.2% in group R. A linear relationship was established between alpha G and sperm content in both groups, but alpha G activities were systematically lower in group P (y = 0.19 x + 64) than in group R (y = 0.30 x + 86). There was no correlation between alpha G and the percent of sperm motility. On the contrary, we found statistically more clinical epididymal abnormalities in cases of decreased alpha G activity than in cases of normal alpha G activity (p less than .01).

Human "spare" embryos, judged unsuitable for freezing because of their poor quality, were cocultured for 5 days on a "Vero" cell layer. These epithelial cells were selected because kidney and genital tract have a common embryologic origin and "Vero" cells are a safe and highly controlled cellular support used for vaccine production. In the control group, the embryos were cultured in culture medium alone (B2 + 15% serum). At the end of the culture, the number of blastocysts was significantly higher in the coculture group: 61% vs. 3%. Moreover, at least half of the blastocysts were expanding and hatching (13/25), with a chronologically normal development. These observations suggest that (1) the coculture system improves human embryonic development; (2) it can rescue early degenerating embryos; (3) beneficial effects of coculture are not strictly genital-tract specific, but rather epithelium dependent. This coculture system could be used for in vitro fertilization to prolong in vitro culture and thus make it possible to transfer embryos at a more appropriate time, to eliminate early-blocked eggs, and to freeze embryos at the blastocyst stage, when freezing procedures are most successful.

A total of 130 transfers of frozen-thawed (F-T) human embryos was carried out after moderate ovarian stimulation with human menopausal gonadotrophin (HMG). Embryos were replaced 3 days after the spontaneous luteinizing hormone (LH) surge or 4 days if ovulation was induced by human chorionic gonadotrophin (HCG). Embryos were thawed a few hours prior to transfer. One-hundred-and-twenty-three transfers were effective and 23 pregnancies were achieved. The rate of ongoing pregnancies per transfer was 17.9% (22/123). The survival rate of embryos originating from cycles stimulated by a combination of an LHRH analogue and HMG in a long protocol (LA-HMG protocol) was significantly lower when compared with the rate of embryos retrieved from clomiphene citrate-HMG (CC-HMG protocol) stimulated cycles (52 versus 67%, P less than 0.05). When fresh embryos originated from cycles stimulated with an LHRH analogue and HMG in a short protocol (SA-HMG protocol), the survival rate was not affected (59 versus 67%, NS). Although the difference was not significant, the ongoing pregnancy rate per transfer according to the three protocols from which the embryos originated seemed to be better with the SA-HMG protocol: 16% with the CC-HMG protocol, 14.5% with the LA-HMG protocol versus 27.6% with the SA-HMG protocol. The success rate was independent of the number of F-T transferred embryos if at least one embryo with 100% intact blastomeres was replaced.

A semiquantitative colorimetric micromethod (APIZYM) was used to study the enzyme profiles of seminal plasma and of spermatozoa. Reactions with 65 different substrates are simultaneously tested in a single specimen. These substrates (principally naphtolic) allow the detection of hydrolytic enzymes (esterases, phosphatases, and peptidases) and of dehydrogenases potentially involved in sperm metabolism and in the process of fertilization. The usual sperm enzymes were regularly observed: C3-C4 esterases, amino acid arylamidases, acrosine, phosphatases, glutamyl transpeptidase, and various osidases. Among the dehydrogenases we observed a striking predominance of the enzymes of the hexose monophosphate shunt and of LDH. Seminal plasma has an enzyme pattern very similar to that of spermatozoa except for the absence of acrosine and of some dehydrogenases. This unexpected similarity is discussed. The gametes from subfertile donors do not at first sight differ in their enzyme pattern from those from fertile donors. Moreover, we found no marked differences between zymograms of seminal plasma from normal, subfertile, or even azoospermic patients. Deep freezing does not modify the hydrolytic enzymes of human sperm either quantitatively of qualitatively, but the dehydrogenases of the hexose monophosphate shunt are adversely affected (60% loss of activity for G 6 PDH, and 30% for 6 PGDH); LDH is not affected. The consequences on fertilizing capacity of frozen semen are discussed.

By the test of experimental deciduomata in the golden Hamster we have built a curve of hormonal antagonism between estradiol benzoate, and progesterone for growthing doses. The mathematical treatment of this curve allows a comparison with the figure of an enzymatic inhibition reaction.

The effects of potassium, calcium and magnesium cations, and albumin on motility of spermatozoa, were tested after 15 min, 4 hr, and 24 hr of incubation using laser Doppler velocimetry. The optimal concentration of K+ was found to be 11 mM; higher values decreased motility more severely than did lower ones, even when K+ was completely removed. Motility was increased in the presence of small amounts of calcium (from 0.8-1.7 mM) but was depressed by magnesium concentrations above 0.8 mM. Moderate concentrations of albumin (0.5-1%) improved motility, but the protecting effect of this compound--leading to a best sperm survival--appeared with higher concentrations (4%). In conclusion we observed that optimal ionic conditions presently found were corresponding well to the tubal environment in preovulatory phase.