Gene expression in human ovarian tissue after xenografting.

Cryobanking and transplantation of ovarian tissue is a promising approach to restore fertility in cancer patients. However, ischemic stress following avascular ovarian cortex grafting is known to induce stromal tissue fibrosis and alterations in follicular development. The aim of the study was to analyze the impact of freeze-thawing and grafting procedures on gene expression in human ovarian tissue. Frozen-thawed ovarian tissue from 14 patients was xenografted for 7 days to nude mice and one ungrafted fragment was used as a control. Immediately after recovery, grafts were processed for RNA extraction and histological analysis. Their expression profile was screened by whole-genome oligonucleotide array (n = 4) and validated by reverse-transcriptase polymerase chain analysis (n = 10). After data filtering, the Limma package was used to build a linear regression model for each gene and to compute its fold change between tissues on Days 0 and 7. After adjusting the P-value by the Sidak method, 84 of the transcripts were significantly altered after 7 days of grafting, including matrix metalloproteinase-9 and -14 and angiogenic factors such as placental growth factor and C-X-C chemokine receptor type 4 (CXCR4). Major biological processes were related to tissue remodeling, including secretory processes, cellular adhesion and response to chemical and hormonal stimuli. Angiopoietin signaling, the interleukin-8 pathway and peroxisome proliferator-activated receptor activation were shown to be differentially regulated. On Day 7, overexpression was confirmed by PCR for interleukin-8, transforming growth factor-beta 1, matrix metalloproteinase-14 and CXCR4, compared with ungrafted controls. In conclusion, new as well as known genes involved in tissue restructuring and angiogenesis were identified and found to play a key role during the first days after human ovarian tissue transplantation. This will facilitate the development of strategies to optimize grafting techniques.

Is transplantation of cryopreserved ovarian tissue from patients with advanced-stage breast cancer safe? A pilot study.

PURPOSE: To assess the safety of reimplantation of cryopreserved ovarian tissue from advanced-stage breast cancer patients. METHODS: Cryopreserved ovarian cortical fragments were obtained from 13 advanced-stage breast cancer patients aged 17-35 years. After thawing, part of the ovarian cortical tissue was grafted to severe combined immunodeficient mice for 6 months. The presence of malignant mammary cells in ovarian tissue was evaluated after thawing as well as after grafting by 1) histology and immunohistochemistry (epithelial membrane antigen, Her2/neu and gross cystic disease fluid protein 15 identification), and 2) detection of the MGB2 gene by qPCR. RESULTS: No malignant cells were evidenced by histology and immunohistochemistry. None of the mice died during the 6-month grafting period, nor developed macroscopically visible masses. MGB2 gene expression was detected by qPCR and confirmed by sequencing in frozen-thawed ovarian tissue in 4 cases and in grafts in 1 case. CONCLUSIONS: This pilot study is the first to evaluate the risk of contamination of cryopreserved ovarian tissue from advanced-stage breast cancer patients by xenotransplantation for 6 months to immunodeficient mice, associated with more conventional screening methods. Our xenografting results are reassuring, but caution needs to be exercised, as MGB2 gene expression was detected in some cases. Larger numbers of ovarian tissue samples from patients with advanced-stage breast cancer are required to confirm our findings before ovarian tissue transplantation can be contemplated in these patients.

Alginate beads as a tool to handle, cryopreserve and culture isolated human primordial/primary follicles.

BACKGROUND: One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME2SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate. METHODS: Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5-15 per group). Follicles were frozen-thawed using 1.4M ME2SO or 1.5M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte. RESULTS: A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME2SO (n=424) or EG (n=259), or used as controls (n=158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME2SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME2SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME2SO groups. CONCLUSIONS: Our results show that 1.4M ME2SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME2SO as a CPA.

Vitrification of non-human primate immature testicular tissue allows maintenance of proliferating spermatogonial cells after xenografting to recipient mice.

This study demonstrates preservation of tissue integrity, maintenance of proliferating spermatogonia and Leydig cell functionality after vitrification and transplantation of non-human primate immature testicular tissue. The objective was to assess the potential of vitrification of non-human primate immature testicular tissue (ITT) in an in vivo xenotransplantation model. Testicular tissue was obtained from one immature rhesus monkey (Macaca mulatta) aged 4 years. Collection and vitrification of testicular tissue, followed by short-term xenografting (3 wks) to nude mice were performed to evaluate and compare vitrified/warmed and fresh tissue. Fresh ungrafted tissue was used for control purposes. Cell density and seminiferous tubule (ST) integrity were assessed by light microscopy. Presence of spermatogonia (SG) (MAGE-A4), proliferation (Ki-67) and Leydig cell (LC) functionality (3ß-hydroxysteroid dehydrogenase; 3ß-HSD) were evaluated by immunohistochemistry (IHC). Qualitative analysis revealed preservation of the histologic characteristics of SG and Sertoli cells (SCs), as well as cell-cell cohesion and cell adhesion to the basement membrane, in both vitrified and fresh grafted tissues. Survival of SG able to proliferate and functional LCs was confirmed by IHC in fresh and vitrified grafts. In conclusion, vitrification appears to be a promising approach, representing an alternative strategy to slow-freezing in the emerging field of ITT cryopreservation and cryobanking.

Inhibition of steroid sulfatase decreases endometriosis in an in vivo murine model.

BACKGROUND: Steroid sulfatase (STS) is involved in estrogen biosynthesis and expressed in eutopic and ectopic endometrium of disease-free and endometriosis patients. The present study was designed to investigate its role in endometriosis development. METHODS: Human endometrial explants were cultured on inserts for 24 h to assess the effectiveness of an STS inhibitor (STS-I), estradiol-3-O-sulfamate (E2MATE), on STS activity in endometrial tissue. Endometriosis was induced in mice, and E2MATE (or vehicle alone) was given orally for 21 days. Plasma estradiol levels were measured, and STS activity was assessed in murine organs (uterus, liver and leukocytes) and in lesions. Lesion number, weight and size (morphometry) were quantified. Lesion STS and progesterone receptor (PR) expression, proliferation and apoptosis rates were determined by immunohistochemistry. RESULTS: In vitro, addition of 1 µM E2MATE to the culture medium resulted in decreased STS activity in endometrial explants (P < 0.001). Treatment of mice with E2MATE (1.0 and 0.5 mg/kg) did not modify plasma estradiol levels, but inhibited STS activity in murine uterus (P < 0.05), liver (P < 0.001) and leukocytes (P < 0.001), as well as in induced lesions (P < 0.05). E2MATE reduced lesion weight (P < 0.01) and size (P < 0.05), but had no impact on proliferation or apoptosis rates, nor STS protein expression. Stromal edema was observed in the uterus of animals treated with E2MATE, but not in the stroma of lesions. Increased PR expression was detected in endometriotic lesions (P < 0.001). CONCLUSIONS: E2MATE was shown to effectively inhibit STS activity in endometrial tissue in vitro. In vivo, E2MATE decreased endometriosis development without affecting systemic estradiol levels. Use of STS-I could therefore be of potential interest in endometriosis treatment.

Endothelial cells are essential for ovarian stromal tissue restructuring after xenotransplantation of isolated ovarian stromal cells.

BACKGROUND: Grafting of isolated follicles represents an approach to prevent the risk of reimplanting malignant cells with cryopreserved ovarian fragments. Optimal conditions and cell types required to sustain human follicular growth need to be identified. To help improve the grafting technique, we investigated whether short-term xenografting of a suspension containing ovarian stromal and endothelial cells without follicles could enhance graft survival and revascularization. METHODS: In human ovary, CD34 selectively labels endothelial cells of blood vessels. A CD34-replete ovarian stromal cell group, including stromal and endothelial cells, was obtained after enzymatic digestion of fresh human ovarian cortex. Magnetic-activated cell sorting was used to establish a CD34-depleted ovarian stromal cell group. Proportions of CD34-positive cells were evaluated by flow cytometry and immunocytochemistry. Cell suspensions were embedded in human plasma clots and grafted (n = 10 for each group, 7 days) to the ovarian bursa of nude mice. Angiogenesis was quantified after human/mouse CD34 immunostaining. RESULTS: CD34-replete grafts had a well-organized and vascularized stromal structure, containing tubular components staining for human CD34 and corresponding to functional vessels, as evidenced by intraluminal red blood cells. CD34-depleted grafts tended to be smaller than CD34-replete grafts and poorly vascularized with central necrosis. Global microvessel density was higher in the CD34-replete than depleted group (337.9 versus 187.3 vessels/mm(2), P < 0.05), with a greater proportion of human vessels (68.02 versus 6.95%, respectively, P < 0.05). CONCLUSIONS: We demonstrated the importance of co-transplanting ovarian endothelial and stromal cells to ensure the formation of a well-vascularized and structured ovarian-like stroma after short-term xenografting, for future application in the transplantation of isolated follicles.

Xenotransplantation of human ovarian tissue to nude mice: comparison between four grafting sites.

BACKGROUND: This study was designed to assess the impact of different ovarian tissue transplantation sites on the follicular pool and ovarian tissue integrity after short-term grafting, since there is no consensus in the literature as to the optimal grafting site in experimental models. METHODS: Frozen-thawed ovarian tissue from eight patients was grafted for 1 or 3 weeks to the peritoneum, inside the ovarian bursa, under the skin and into the muscle of 16 nude mice. Assessment of follicular density and follicle classification was carried out by histological analysis. Proliferative activity was evidenced by immunostaining with anti-Ki-67 antibodies, and fibrotic areas were analyzed by morphometry on histological slides. RESULTS: One week post-transplantation, the proportion of Ki-67-positive primordial follicles was higher (20-42%) than in controls (1.7%), demonstrating follicular activation in all four sites. Despite this activation, primordial follicles were still found 3 weeks post-grafting, (34.1-66.9% of the follicle population), most of them quiescent, as indicated by the absence of Ki-67 immunostaining. Cryopreservation and grafting resulted in extensive fibrosis in the stroma. This fibrosis was significantly less pronounced in intramuscular (IM) grafts, representing 18.8% of the surface versus 44.7-60.5% for other sites, after 3 weeks of grafting. CONCLUSIONS: All four grafting sites equally supported early follicular growth and preserved some quiescent follicles after short-term frozen-thawed human ovarian tissue transplantation. The extensive fibrosis observed does not appear to have a major impact on early follicle development, but its long-term effects must be investigated. The graft environment may be implicated in the preservation of the stroma, as suggested by a lower degree of fibrosis in the IM site.

Short-term exposure to hydrogen peroxide during oocyte maturation improves bovine embryo development.

Recent studies have shown that short-term exposure of oocytes to a stressor such as hydrostatic pressure or osmotic stress might induce stress tolerance in embryos. The aim of the present study was to investigate the consequences of short-term hydrogen peroxide (H(2)O(2)) exposure to bovine in vitro matured cumulus-oocyte complexes (COCs) on subsequent preimplantation embryo development and apoptosis. In the first experiment, mature COCs were incubated in H(2)O(2) at concentrations ranging between 0.01 and 100 micromol/l, and subsequently fertilized and cultured. Oocyte incubation with 50-100 micromol/l of H(2)O(2) resulted in a significantly higher blastocyst yield (47.3%) in comparison with control medium (31.8%), while apoptotic cell ratio was inversely related with H(2)O(2) concentration. In the second experiment, we showed that the stress tolerance after H(2)O(2) exposure was not mediated by increased glutathione content in treated oocytes nor by enhanced fertilization or penetration. Further research should concentrate on the potential role of players that have been associated with stress tolerance in somatic cell lines.

Expression of eicosanoid biosynthetic and catabolic enzymes in peritoneal endometriosis.

BACKGROUND: Increased peritoneal eicosanoid concentrations have been reported in endometriosis patients and might be important in disease-associated pain and inflammation. Here, we evaluated the expression of key biosynthetic and catabolic enzymes involved in this abnormal eicosanoid production in peritoneal macrophages and endometriotic lesions. METHODS: Peritoneal macrophages, endometriotic lesions and matched eutopic endometrium were collected from endometriosis patients (n = 40). Peritoneal macrophages and eutopic endometrium samples were also collected from disease-free women (n = 25). Expression of type IIA secretory phospholipase A(2) (sPLA(2)-IIA), cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and 5-lipoxygenase (5-LO) was quantified by real-time PCR, and these five key enzymes were localized by immunohistochemistry. RESULTS: sPLA(2)-IIA, COX-2 and mPGES-1 mRNA was significantly increased in peritoneal macrophages of endometriosis patients compared with controls (P = 0.006, P = 0.016 and P = 0.025, respectively). In endometriosis patients, sPLA(2)-IIA, mPGES-1 and 15-PGDH mRNA was significantly enhanced in peritoneal lesions compared with matched eutopic endometrium (P < 0.001, P < 0.001 and P = 0.005, respectively). In eutopic endometrium, a significant decrease in 15-PGDH mRNA was found in the endometriosis group compared with controls (P = 0.023). Finally, sPLA(2)-IIA, COX-2, mPGES-1 and 15-PGDH immunostaining was found mainly in endometrial glands, whereas 5-LO was distributed throughout the glands and stroma. CONCLUSIONS: Our study highlights an imbalance between eicosanoid biosynthesis and degradation in endometriosis patients. Both peritoneal macrophages and endometriotic lesions may be involved. Research into new molecules inhibiting biosynthetic enzymes (such as sPLA(2)-IIA and mPGES-1) and/or activating catabolic enzymes (such as 15-PGDH) may prove to be a major field of investigation in the development of targeted medical therapies.

Absence of aromatase protein and mRNA expression in endometriosis.

BACKGROUND: Aromatase has been reported to be involved in estrogen biosynthesis and expressed in eutopic and ectopic endometrium of endometriosis patients. The objective of the present study was to investigate its expression and localization in three distinct types of endometriosis. METHODS: Human peritoneal, ovarian and rectovaginal endometriotic lesions and matched eutopic endometrium were collected from patients during laparoscopy. Aromatase protein localization (immunohistochemistry, n = 63) and mRNA expression [quantitative polymerase chain reaction (Q-PCR), n = 64] were assessed. RESULTS: No aromatase protein was detected by immunohistochemistry in either the glandular or stromal compartment of endometriotic lesions or eutopic endometrium, while it was strong in placental syncytiotrophoblasts, granulosa and internal theca cells from pre-ovulatory follicles, and luteal cells from corpus luteum. By Q-PCR, low but discernible levels of aromatase expression were found in endometriomas, probably due to follicular expression. Transcripts for aromatase were barely detectable in only a few peritoneal and rectovaginal endometriotic lesions, and a few eutopic endometrium samples, probably due to contaminating surrounding tissues (adipose tissue, intact peritoneum). CONCLUSIONS: Unlike previous studies, we observed no aromatase protein in any of the endometriosis types, and barely detectable aromatase mRNA expression, suggesting that locally produced aromatase (within endometriotic lesions) may be less implicated in endometriosis development than previously postulated. Potential factors responsible for these discrepancies are discussed.

Potential involvement of iron in the pathogenesis of peritoneal endometriosis.

The aim of this study is to review the current literature associating endometriosis with iron and to discuss the potential causes and consequences of iron overload in the pelvic cavity. Indeed, iron is essential for all living organisms. However, excess iron can result in toxicity and is associated with pathological disorders. In endometriosis patients, iron overload has been demonstrated in the different components of the peritoneal cavity (peritoneal fluid, endometriotic lesions, peritoneum and macrophages). Animal models allow us to gather essential information on the origin, metabolism and effect of iron overload in endometriosis, which may originate from erythrocytes carried into the pelvic cavity mainly by retrograde menstruation. Peritoneal macrophages play an important role in the degradation of these erythrocytes and in subsequent peritoneal iron metabolism. Iron overload could affect a wide range of mechanisms involved in endometriosis development, such as oxidative stress or lesion proliferation. In conclusion, excess iron accumulation can result in toxicity and may be one of the factors contributing to the development of endometriosis. Treatment with an iron chelator could thus be beneficial in endometriosis patients to prevent iron overload in the pelvic cavity, thereby diminishing its deleterious effect.

Identification of novel antigens in blood vessels in rectovaginal endometriosis.

To identify specific markers of rectovaginal endometriotic nodule vasculature, highly enriched preparations of vascular endothelial cells and pericytes were obtained from endometriotic nodules and control endometrial and myometrial tissue by laser capture microdissection (LCM), and gene expression profiles were screened by microarray analysis. Of the 18 400 transcripts on the arrays, 734 were significantly overexpressed in vessels from fibromuscular tissue and 923 in vessels from stromal tissue of endometriotic nodules, compared with vessels dissected from control tissues. The most frequently expressed transcripts included known endothelial cell-associated genes, as well as transcripts with little or no previous association with vascular cells. The higher expression in blood vessels was further corroborated by immunohistochemical staining of six potential markers, five of which showed strong expression in pericytes. The most promising marker was matrix Gla protein, which was found to be present in both glandular epithelial cells and vascular endothelial cells of endometriotic lesions, although it was barely expressed at all in normal endometrium. LCM, combined with microarray analysis, constitutes a powerful tool for mapping the transcriptome of vascular cells. After immunohistochemical validation, markers of vascular endothelial and perivascular cells from endometriotic nodules could be identified, which may provide targets to improve early diagnosis or to selectively deliver therapeutic agents.

Estrogen metabolizing enzymes in endometrium and endometriosis.

BACKGROUND: Estradiol (E(2)) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent. METHODS: In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17beta-hydroxysteroid dehydrogenases (17beta-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)] in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures. RESULTS: Aromatase and 17beta-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17beta-HSD, EST and STS were readily detectable. Only 17beta-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17beta-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies. CONCLUSIONS: In endometriosis lesions, the balance is tilted in favor of enzymes producing E(2). This is due to a suppression of types 2 and 4 17beta-HSD, and an increased expression of aromatase and type 1 17beta-HSD in ectopic endometrium.

Allograft of ovarian cortex between two genetically non-identical sisters: case report.

Aggressive chemotherapy and radiotherapy generally result in the loss of both endocrine and reproductive functions. In 1990, a woman aged 20 years, presenting with beta-thalassemia major, underwent chemotherapy (busulfan and cyclophosphamide) and total body irradiation (TBI) before bone marrow transplantation (BMT), the donor being her 17-year-old HLA-compatible sister. The treatment resulted in premature ovarian failure. In 2006, after excision of ovarian cortical fragments from the HLA-compatible sister, these fragments were immediately sutured to the ovarian medulla of the patient. Both procedures were performed by laparoscopy. Six months after reimplantation, vaginal ultrasonography and hormone concentrations indicated recovery of ovarian secretion and function. From 6 to 11 months, the patient experienced menstrual bleeding and the development of a follicle concomitant with high estradiol levels. Eleven months after reimplantation, two follicles were detected and punctured under vaginal ultrasonographic control. Two mature oocytes were retrieved and inseminated by ICSI. Two embryos (2- and 3-cell) were obtained. Allotransplantation of fresh ovarian tissue was laparoscopically performed between two genetically non-identical sisters. Restoration of ovarian function was achieved after six months. Oocyte retrieval and embryo development were demonstrated.

Restoration of ovarian function after orthotopic (intraovarian and periovarian) transplantation of cryopreserved ovarian tissue in a woman treated by bone marrow transplantation for sickle cell anaemia: case report.

Ovarian function after orthotopic transplantation of cryopreserved ovarian tissue has been restored in women with malignant disease. Here the techniques are adapted for a non-cancer patient. In 1999, right oophorectomy was performed in a 21 year old woman before chemotherapy, prior to bone marrow transplantation. Ovarian cortex was frozen, according to a strict protocol. After thawing, ovarian cortex was reimplanted into the ovary and in a peritoneal window close to the ovary in 2004. Four-and-a-half months after reimplantation, LH, FSH, 17beta-estradiol and progesterone levels, as well as ultrasonography, demonstrated the presence of an ovulatory cycle. After this cycle, the patient experienced two other ovulatory cycles, evidenced by FSH and 17beta-estradiol concentrations, as well as ultrasound demonstration of a follicle. Follicular development was clearly observed in both the intraovarian site (1st and 2nd cycle) and the peritoneal window (3rd cycle). Restoration of endocrine ovarian function occurred after ovarian cortical strips, biopsied and cryopreserved before chemotherapy, were reimplanted into the ovary itself and a periovarian peritoneal window.

Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.

BACKGROUND: The lifesaving treatment endured by cancer patients leads, in many women, to early menopause and subsequent infertility. In clinical situations for which chemotherapy needs to be started, ovarian tissue cryopreservation looks to be a promising option to restore fertility. In 1997, biopsy samples of ovarian cortex were taken from a woman with stage IV Hodgkin's lymphoma and cryopreserved before chemotherapy was initiated. After her cancer treatment, the patient had premature ovarian failure. METHODS: In 2003, after freeze-thawing, orthotopic autotransplantation of ovarian cortical tissue was done by laparoscopy. FINDINGS: 5 months after reimplantation, basal body temperature, menstrual cycles, vaginal ultrasonography, and hormone concentrations indicated recovery of regular ovulatory cycles. Laparoscopy at 5 months confirmed the ultrasonographic data and showed the presence of a follicle at the site of reimplantation, clearly situated outside the ovaries, both of which appeared atrophic. From 5 to 9 months, the patient had menstrual bleeding and development of a follicle or corpus luteum with every cycle. 11 months after reimplantation, human chorionic gonadotrophin concentrations and vaginal echography confirmed a viable intrauterine pregnancy, which has resulted in a livebirth. INTERPRETATION: We have described a livebirth after orthotopic autotransplantation of cryopreserved ovarian tissue. Our findings suggest that cryopreservation of ovarian tissue should be offered to all young women diagnosed with cancer.

Comparison of G1.2/G2.2 and Sydney IVF cleavage/blastocyst sequential media for the culture of human embryos: a prospective, randomized, comparative study.

OBJECTIVE: To compare two commercially available sequential media, G1.2/G2.2 and Sydney IVF cleavage/blastocyst media, as supports for human embryo culture. DESIGN: Prospective randomized study. SETTING: University-based IVF clinic. PATIENT(S): Two hundred forty-nine patients undergoing IVF treatment for the first or second time, randomly allocated at the time of oocyte retrieval, to either culture in G1.2/G2.2 or Sydney IVF media. INTERVENTION(S): Oocyte recovery, IVF or intracytoplasmic sperm injection, embryo culture, transfer on day 3 or day 5/6. MAIN OUTCOME MEASURE(S): Developmental stage on day 3, blastocyst rate, pregnancy outcome as assessed by beta hCG positive test, implantation rates, and ongoing pregnancies. RESULT(S): Embryos cultured in G1.2/G2.2 media displayed a faster kinetics of cleavage, compaction, blastulation, and hatching, but a lower day 3 embryo quality than those grown in Sydney IVF media. For patients with at least five embryos, G1.2/G2.2 media yielded higher implantation rates (26.2%) in our day 3 embryo transfer program when compared to Sydney IVF medium (15.5%), whereas similar implantation rates were obtained for day 5/6 embryo transfer for both media (43.1% and 36.1%, respectively). CONCLUSION(S): In our day 3 embryo transfer program, G1.2/G2.2 media were superior to Sydney IVF media, whereas both media yielded similar outcomes in our blastocyst transfer program.

Biochemical analysis of the sperm and infertility.

The 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 alpha-glucosidase) as a screening method in male infertility investigation.

Expression of Cu/Zn and Mn superoxide dismutases during bovine embryo development: influence of in vitro culture.

Temporal pattern of expression of Cu/Zn and Mn superoxide dismutases (SODs) was investigated in bovine oocytes and embryos produced in vitro in two different culture conditions and in vivo after superovulation. SODs were examined at a transcriptional level in single oocytes and embryos by reverse transcriptase-polymerase chain reaction (RT-PCR) and, at a protein level, by Western blotting on pools of embryos. mRNA encoding Cu/Zn SOD were detected in in vitro bovine embryos throughout preattachment development as well as in in vivo derived morulae and blastocysts. Transcripts for Mn SOD gene were detected in most immature and in vitro matured oocytes as well as in some zygotes and 5- to 8-cell embryos while no transcript was found at the 9-to 16-cell stage in both culture conditions. In vitro embryonic expression of Mn SOD was detected earlier in the presence of serum. Half of the morulae showed the transcript if cultured with 5% serum while none without serum. At the blastocyst stage Mn SOD could be detected independently of culture conditions. For in vivo-derived embryos Mn SOD transcripts were detected both in morulae and blastocysts. Immunoblotting analyses revealed that Cu/Zn SOD and Mn SOD were also present at a protein level in in vitro-derived zygotes and blastocysts. Together these data demonstrate, for the first time, that Mn SOD is transcribed and that Cu/Zn and Mn SOD proteins are expressed in preimplantation bovine embryos. Finally, they suggest that Mn SOD transcription is altered by in vitro culture conditions.

Pulsatile activity and hatching of in vitro produced cow blastocysts: effects of serum supplementation.

The effect of serum added to a modified SOF medium on pulsatile activity and hatching of in vitro produced cow blastocysts was investigated by time-lapse cinematography. Embryos were generated from abattoir material and cultured in mSOF without serum or with 10% FCS added at 42h pi. Addition of serum significantly increases pulsatile activity before zona rupture and reduces the time of hatching. Pulsatile activity does not seem to be involved in the hatching process.