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Objective Detection of nerve fibers in endometrial biopsies was recently proposed as a noninvasive diagnostic tool for endometriosis. However, their occurrence in the functional layer of endometrium still remains controversial. Nerve fibers were found to be present in endometriotic lesions themselves, which may account for some of the pain experienced by patients, but their origin is not clear. The objective of the present study was to reevaluate the presence of nerve fibers in endometrium and in different types of endometriotic lesions.Patients and Methods Nerve fiber density (PGP9.5 immunohistochemical analysis), unmyelinated nerve fiber presence (neurofilament immunohistochemical detection) and nerve growth factor expression were evaluated in endometrial (disease free: n = 20; endometriotic: n = 26) and endometriotic (peritoneal lesions: n = 11; ovarian lesions: n = 16; rectovaginal lesions: n = 27) samples.Results Endometrial biopsies were found to be mostly negative for nerve fibers. Nerve fiber density was higher in deep nodular lesions than in peritoneal (p<0.01) or ovarian (p<0.001) lesions. Around 30% of PGP9.5-positive nerve fibers were confirmed by neurofilament staining. Nerve growth factor expression was detected at higher levels in the stroma of deep-infiltrating lesions (p<0.05).Conclusions No nerve fibers were detected in endometrial biopsies (from healthy or endometriosis patients). However, nerve fibers were detected in endometriotic lesions. Most of them were found to be unmyelinated, suggesting they could be implicated in pain. Deep nodular lesions may be more neuroattractive through the action of nerve growth factor.

In this study, we developed an original and reproducible quantitative in vitro model of endometrial cell adhesion to peritoneal mesothelial cells in order to better assess the impact of pro-inflammatory cytokines on early-stage endometriosis development. We demonstrated that pre-treatment with TNF-α and IL-1β does not promote endometrial stromal cell adhesion to peritoneal mesothelial cells.

Ovarian tissue cryopreservation is currently proposed to young cancer patients to preserve their fertility before radiochemotherapy. The potential risk is that the tissue might harbor malignant cells that could induce disease recurrence. We therefore decided to evaluate the presence of leukemic cells in cryopreserved ovarian tissue from 18 leukemic patients: 6 with chronic myeloid leukemia (CML) and 12 with acute lymphoblastic leukemia (ALL). In each case, histology, quantitative reverse-transcribed polymerase chain reaction (RT-PCR) and long-term (6 months) xenografting to immunodeficient mice were used. Histology did not identify any malignant cells in the ovarian tissue. By quantitative RT-PCR, 2 of 6 CML patients were positive for BCR-ABL in their ovarian tissue. Among the 12 ALL patients, 7 of the 10 with available molecular markers showed positive leukemic markers in their ovarian tissue (translocations or rearrangement genes). Four mice grafted with ovarian tissue from ALL patients developed intraperitoneal leukemic masses. In conclusion, this study demonstrates, by quantitative RT-PCR, ovarian contamination by malignant cells in acute as well as chronic leukemia, whereas histology fails to do so. Moreover, chemotherapy before ovarian cryopreservation does not exclude malignant contamination. Finally, reimplantation of cryopreserved ovarian tissue from ALL and CML patients puts them at risk of disease recurrence.

Advances in the diagnosis and treatment of childhood, adolescent and adult cancer have greatly increased the life expectancy of premenopausal women with cancer.

Purpose Ectopic endometrial tissue is biochemically and functionally distinct from eutopic endometrium, including differences in receptivity to steroids and invasive potential. For this reason, using ectopic endometrial tissue to induce endometriosis in animal models may be of interest to understand better the pathogenesis of this disease. The aim of this study was to evaluate the potential of human pelvic endometriotic lesions (specifically red lesions) to implant into the peritoneal cavity of nude mice.Methods Human peritoneal endometriotic tissue (red lesions) was injected into the pelvic cavity of nude mice. The mice were euthanized after 5 or 14 days, lesions were recovered, and histological analysis was performed. Proliferative activity and neoangiogenesis of lesions were assessed after Ki67 and CD34 immunohistological staining, respectively.Results After 5 and 14 days, endometriotic tissue was found to have implanted onto intact mesothelium and reorganized into structured glands and stroma, forming endometriosis-like lesions. These lesions showed considerable proliferative activity and effective neoangiogenesis.Conclusion This model represents a promising tool to study in vivo implantation of human ectopic endometrium, which is dissimilar to eutopic endometrium. Moreover, this study shows that glands and stroma detached from red endometriotic lesions are able to implant in other peritoneal sites, as in a metastatic process.

Peritoneal endometriosis is a chronic pelvic inflammatory disease, characterized by increased numbers of peritoneal macrophages and their secreted products such as cytokines, growth and angiogenic factors in peritoneal fluid. Inflammation plays a major role in pain and infertility associated with endometriosis, but is also extensively involved in the molecular and cellular processes that lead to peritoneal endometriotic lesion development. Several inflammatory mediators have therefore been studied in the context of endometriosis over the last few years.The aim of this review is to focus on three that have been clearly implicated in the pathogenesis of endometriosis and may be linked: peritoneal iron metabolism, nuclear factor-kappa B (NF-κB) activation, and prostaglandin biosynthesis.Peritoneal iron overload has been conclusively demonstrated in endometriosis patients and may induce oxidative stress in the peritoneal cavity. Oxidative stress and proinflammatory cytokines are well known to be potent activators of the NF-κB pathway, which has recently been implicated in peritoneal endometriosis. Induced NF-κB activation leads to expression of numerous proinflammatory genes such as cytokines, which may provide positive feedback to the pathway, self-perpetuating the inflammatory response. Other important NF-κB-regulated molecules are prostaglandin biosynthesis enzymes, and cyclooxygenase-2 (COX-2) in particular. Increased concentrations of prostaglandins have been evidenced in the peritoneal fluid of endometriosis patients and COX-2 inhibitors have proved to be effective in ‘in vitro’ and ‘in vivo’ experimental models.In the light of available data collected from patient biopsies, as well as ‘in vitro’ and ‘in vivo’ studies, the respective implication and potential molecular association of iron, NF-κB and prostaglandins in the pathogenesis of endometriosis are discussed. The key role of peritoneal macrophages is emphasized and potential therapeutic targets are examined.

The review covers current options for ovarian tissue cryopreservation and transplantation and provides a systematic review of the existing literature from the last 10 years, taking into account all previously published reviews on the subject. The different cryopreservation options available for fertility preservation in cancer patients are embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation. The choice depends on various parameters: the type and timing of chemotherapy, the type of cancer, the patient’s age and the partner status. The different options and their results are discussed, as well as their putative indications and efficacy. The review concludes that advances in reproductive technology have made fertility preservation techniques a real possibility for patients whose gonadal function is threatened by premature menopause, or by treatments such as radiotherapy, chemotherapy or surgical castration.

The purpose of this review is to investigate recent advances in xenografting, as well as in orthotopic and heterotopic autotransplantation of human cryopreserved ovarian tissue.

To review the concept of typical, subtle and invisible endometriosis and analyze the evolution and progression of the disease, as well as some factors possibly involved in the pathogenesis.

Sucrose (0.3 M) was used to cause artificial compaction of the embryonic cell mass of in vitro produced bovine embryos to facilitate morphological evaluation. Embryos were produced using routine in vitro maturation (IVM) and fertilization (IVF) techniques. The time necessary to induce shrinkage in 0.3 M sucrose to 75% of the original volume of Day 5 morulae was found to be less than l min, and 95% of the volume was regained in PBS after 2.5 min. No detrimental effect was observed after a 5- to 10-min sucrose treatment on subsequent blastocyst formation at Days 6 and 7 (P 0.05). Furthermore, no significant differences were observed in the total number of cells, or in the mitotic and pycnotic cell index of blastocysts in different treatment groups. Agreement among 7 evaluators grading 40 Day 6 embryos was examined using the kappa coefficient of agreement (κ). Overall agreement among evaluators for classification of quality grade was poor (48.2 %, κ = 0.31) for embryos evaluated in PBS, but the rate improved when the same embryos were scored in sucrose (62.5 %, κ = 0.49). Evaluating less compact in vitro produced bovine morulae in sucrose increases agreement among evaluators, since embryos in sucrose mimick the appearance of in vivo produced embryos. Thus, we conclude that scoring in vitro produced embryos in sucrose improves agreement among evaluators.

The effects of fetal calf serum (FCS) or serum fractions on the development of bovine embryos was investigated. Bovine zygotes were produced in vitroand were cultured in a semi-defined culture medium (mSOF). In the first experiment, blastocysts produced in mSOF supplemented with 10% whole heat-treated FCS or desalted FCS appeared about 1 day earlier, their proportion was significantly (P< 0.05) higher (whole: 30%, desalted: 29%) and they had significantly (P< 0.05) more cells at day 8 (119 cells, 127 cells) than did blastocysts produced in mSOF without any supplement (16%, 98 cells) or mSOF supplemented with a glucose concentration equivalent to that of serum (15%, 88 cells). Our results indicate that high molecular mass components (>5 kDa) of serum are responsible for the effects of FCS on the kinetics of development, on the percentage of blastocysts obtained and the total number of cells in blastocysts. A further analysis using time-lapse microcinematography showed that the acceleration of development induced by serum occurred between the 9–16-cell and morula stages. Finally, in an experiment designed to analyse by microcinematography the effect of the addition of FCS using semen from a different bull to inseminate the oocytes, a different batch of serum and adding mSOF at a different time (42 h after insemination), acceleration was similarly observed between these two stages. Our microcinematographic studies demonstrate that the addition of FCS at two developmental stages (three–four-cell and five–eight-cell) before the 8–16-cell stage accelerates development just after this critical blocking stage.

The objective of this study was to compare the development and metabolic activity of cattle embryos co-cultured with bovine oviductal cells or cultured in serum-free medium previously conditioned by bovine oviductal cells. Zygotes were produced by in vitrofertilization of oocytes from bovine ovaries obtained from an abattoir. Development to the four-cell stage occurred by 48 h after fertilization in both culture systems, but co-cultured embryos reached the 16-cell stage by 96 h, whereas those cultured in conditioned medium did not do so until 24 h later. Similarly, the morula and blastocyst stages were reached 24 h earlier in co-culture than in conditioned medium. There were significantly more cells in the blastocysts from co-culture (96.8 ± 6.1 versus 56.7 ± 3.3; P≤ 0.0001). The metabolism of glutamine did not differ between embryos cultured in the two systems, but the metabolism of glucose was significantly greater in embryos cultured in conditioned medium. The first significant increase in glucose metabolism occurred between the four-cell and the 16-cell stages in embryos cultured in conditioned medium, but occurred between the 16-cell and morula stages in the co-cultured embryos, such that the glucose metabolism was significantly greater at the 16-cell stage in embryos cultured in conditioned medium compared with co-cultured embryos (6.5 ±1.0 versus 1.5 ± 0.4 pmol per embryo per 3 h, P≤ 0.0001). The concentration of glucose was significantly less, and that of lactate significantly greater, in co-culture medium than in conditioned medium. The results suggest that the activity of enzymes involved in glucose transport or metabolism in the early cattle embryo can be affected by the prior culture conditions, and that a high rate of glucose metabolism may be unfavourable for development.

Epidermal growth factor (EGF) has been shown to have a positive effect during in vitro maturation (IVM) and has been reported in follicular fluid at levels capable of stimulating meiosis in a variety of species. The aim of the present work was to study the effect on subsequent development of EGF present in defined medium during bovine 1) oocyte maturation or 2) embryo culture. The presence of EGF during IVM, irrespective of concentration (1, 10, 100 ng/ml), stimulated cumulus expansion and significantly increased the proportion of oocytes attaining metaphase II, the rate of cleavage, and the proportion of embryos reaching the 5- to 8-cell stage at 72 h postinsemination. Blastocyst rates on Days 7 and 9 were also significantly improved for oocytes matured in the presence of EGF (10% vs. 18–24% on Day 7 and 21% vs. 31–32% on Day 9, for Tissue Culture Medium 199 [M199] and M199 + EGF, respectively). The presence of fetal calf serum (FCS) during IVM resulted in similarly elevated rates of development. There was no cumulative effect when EGF and FCS were present together during IVM. The presence of EGF also altered the pattern of proteins neosynthesized during maturation. The maturationpromoting effect of EGF was evident for denuded oocytes also, suggesting that EGF may act, at least in part, directly on the oocyte. Immunofluorescence studies revealed the EGF receptor on immature cumulus-oocyte complexes. When present during postfertilization culture in defined medium (synthetic oviduct fluid), EGF stimulated development in comparison to that of the control but could not replace serum. The results suggest a physiological role for EGF in the regulation of bovine oocyte maturation and development.

A survey of calving outcome from embryos produced in vitro by three laboratories under different culture conditions is presented. A total of 572 embryos were transferred after culture and 170 calves were born: 9 calves from 47 (19%) embryos produced in coculture with bovine oviduct cells (BOCC) or medium conditioned by these cells (BOCMS +); 5 from 16 (31%) embryos produced in serum free oviduct conditioned medium (BOCMS−); 8 from 24 (33%) embryos derived from BRL conditioned medium (BRLCM−); 0 from 9 embryos cultured in SOF (20% O2); 132 from 427 (31%) cocultured with granulosa cells (GCC); 16 from 49 (33%) embryos cultured in SOF (5% O2). Frozen embryos (n= 120) were transferred soon after thawing and 25 calves were obtained: 11 from 59 (19%) embryos in the first condition (BOCC); 6 from 19 (32%) in the second condition (BOCMS +); 6 from 34 (18%) in the third (BOCMS−) and 2 from 8 (25%) in the fourth condition (BRLCMS−). Gestation length and birth weight were in the normal range except for 5 calves that were exceptionally large. Sex ratio was in favour of males. Neo and perinatal mortality ranged from 0 to 19%. Three cases of malformations were observed. Growth rate was normal.

The concentrations of non chloroplastic NAD+, NADP+, NADH and NADPH were followed during the development of the unicellular green alga Acetabularia. When Acetabularia enters the rapid growth phase, a slight increase in NAD+ level is observed, and a slight decrease in both NADH and NADPH levels. In contrast, a huge decrease in both NAD+ and NADP+ concentrations take place at the time caps are initiated and develop, together with a slight increase in both reduced pyridine nucleotides concentrations. This increase has been confirmed and extended from the primordium stage to the cysts bearing caps: NADH and NADPH steadily increase in concentration.The circadian rhythms of the four pyridine nucleotide concentrations have also been studied in the non chloroplastic fraction of Acetabularia. Both NAD+ and NADP+ concentrations oscillate in phase according to a biphasic mode. The first peak occurs at 3 h (CT) and the second one at 15h, whereas the troughs are at 6 and 22 h. The rhythms are maintained in constant dark, in phase with that of light-dark algae (with the possible delay of the minima). The reduced pyridine nucleotides concentrations are very much lower than those of the oxidized forms; this fact, together with the large fluctuations make any conclusion as for the CR of the reduced forms uncertain.

AbstractThe clinical value of biochemical analysis of sperm is still unclear. In this study, we evaluated the potential of several biochemical markers in the seminal plasma (zinc, citrate, acid phosphatase, fructose and neutral α-glucosidase) as a screening method in male infertility investigation.