Comparison of Morphological and Digital-Assisted Analysis for BCL6 Endometrial Expression in Women with Endometriosis.

BCL6 (B-cell lymphoma 6) is a proto-oncogene and transcriptional repressor initially described as being involved in B-cell lymphoma. Recently, this factor has been identified as a promising tissue biomarker which could be used to diagnose women affected by endometriosis. Previous studies used HSCORE for BCL6 staining quantification in the endometrium. However, this semi-quantitative technique of analysis has some limitations, including a lack of objectivity, robustness, and reproducibility that may lead to intra- and inter-observer variability. Our main goal was to develop an original computer-assisted method to quantify BCL6 staining from whole-slide images reliably. In order to test the efficiency of our new digital method of quantification, we compared endometrial BCL6 expression between fertile and infertile women without or with different stages of endometriosis by using the widely used HSCORE analysis and our new automatic digital image analysis. We find a higher expression of BCL6 in the endometrium of infertile women with endometriosis and women with stage IV endometriosis. Furthermore, we demonstrate a significant correlation between the two types of independent measurements, indicating the robustness of results and also the reliability of our computer-assisted method for BCL6 quantification. In conclusion, our work, by using this original computer-assisted method, enables BCL6 quantification more objectively, reliably, robustly, and promptly compared to HSCORE analysis.

Oocyte Cryopreservation in Patients with Endometriosis: Current Knowledge and Number Needed to Treat.

The rise of oocytes cryopreservation (OOC) in assisted reproductive techniques allows fertility preservation (FP) in an increasing number of indications. Endometriosis, a highly prevalent disease, potentially impairing ovarian reserve, seems, therefore, an interesting indication for it. The purpose of this study is to summarize the available evidence concerning FP by OOC in women with endometriosis and to calculate the number needed to treat (NNT). In total, 272 articles related to this topic were identified in PubMed. Eight studies were eligible for the review. In order to shed some light, a SWOT analysis was performed and the argument pros and cons were developed. The NNT calculated of OOC was 16, meaning that 16 women need to perform an OOC for one of them to have a child that she would not have had without this technique. In conclusion, OOC must be discussed with patients who suffer from endometriosis since it is an effective technique of FP, which can allow these patients to succeed a pregnancy that they otherwise would not have achieved. Nevertheless, it should not be performed in all patients as there is still a lack of robust socio-economic and risk-benefit data.

Preservation of female fertility: The current therapeutic strategy.

BACKGROUND: Preservation of female fertility is a relatively new field in medicine that has grown very rapidly in recent decades. At the beginning, embryo freezing remained the most effective technique. Thereafter, cryopreservation of oocytes and ovarian tissue was considered a secure tool in human fertility preservation. Storage of cortical ovarian tissue is moreover relevant for children, prepubertal girls, and adult patients who cannot benefit from cryopreservation of oocytes. OBJECTIVE: To analyze and review recent and relevant scientific literature on medical and social reasons for preservation of fertility. METHODS: The review was conducted based on articles identified from PubMed databases using keywords. MAIN RESULTS: Oocyte vitrification allows women to preserve their fertility without the need for fertilization. Nowadays, thousands of healthy children have been born from this procedure. Occurrence of pregnancy depends on two main factors: the number of mature oocytes in storage and the age of the patient at the time of vitrification. Numerous adaptations have been developed to suit the ovarian stimulation regiments to patients with cancer. In young prepubertal girls, freezing of ovarian tissue remains the best and only option. CONCLUSION: Oocyte vitrification therefore appears to be the gold standard technique of preserving fertility in young women.

Comparison between paraffin and mineral oil covering on early human embryo culture: a prospective randomized study.

The oil overlay in microdrop culture systems prevents medium evaporation, helps to maintain appropriate pH and osmotic conditions and protects from microbial contamination. In the present study, we prospectively compared covering by Ovoil™, a paraffin oil, and LiteOil®, a mineral oil, on the in vitro development of human embryos and their suitability for transfer/freezing at day 3 and live birth rate. One hundred and one patients undergoing in vitro fertilization (IVF) treatment by  intracytoplasmic sperm injection (ICSI) were enrolled in our study. After ICSI, 1237 oocytes were 1:1 randomly allocated into 2 groups according to the type of overlaying oil: Ovoil™ (616 oocytes) or LiteOil® (621 oocytes). Fertilization rate was assessed around 18 hours post-insemination (hpi) and embryos were checked for early cleavage at 25 hpi. Embryo morphology was recorded on days 2 and 3. A total of 437 (Ovoil™) and 438 day 3 embryos (LiteOil®) were analyzed. There were no differences between the two groups in terms of fertilization rate and occurrence of early cleavage. The proportion of top quality embryos (41.7% vs. 41.2%) and the final utilization rates (92.2% vs. 92.0%) were similar in Ovoil and LiteOil groups, respectively, at day 3. Live birth rate per transfer was essentially the same with Ovoil™ overlay (26.9%) when compared to LiteOil® (26.2%). Live birth rate in patients who simultaneously received  embryos from both overlay types was 17.2%. Despite the different characteristics of these two oils regarding hydrocarbon saturation, packing and temperature storage, Ovoil™ and LiteOil® can be used in parallel in the same IVF protocol. Abbreviations: ART: assisted reproductive technologies; hpi: hours post-insemination; hSA: human serum albumin; HTF: human tubal fluid; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; MII: metaphase II; MEA: mouse embryo assay; RT: room temperature.

Evaluation of Z-VAD-FMK as an anti-apoptotic drug to prevent granulosa cell apoptosis and follicular death after human ovarian tissue transplantation.

PURPOSE: To evaluate the efficiency of ovarian tissue treatment with Z-VAD-FMK, a broad-spectrum caspase inhibitor, to prevent follicle loss induced by ischemia/reperfusion injury after transplantation. METHODS: In vitro, granulosa cells were exposed to hypoxic conditions, reproducing early ischemia after ovarian tissue transplantation, and treated with Z-VAD-FMK (50 µM). In vivo, cryopreserved human ovarian fragments (n = 39) were embedded in a collagen matrix containing or not Z-VAD-FMK (50 µM) and xenotransplanted on SCID mice ovaries for 3 days or 3 weeks. RESULTS: In vitro, Z-VAD-FMK maintained the metabolic activity of granulosa cells, reduced HGL5 cell death, and decreased PARP cleavage. In vivo, no improvement of follicular pool and global tissue preservation was observed with Z-VAD-FMK in ovarian tissue recovered 3-days post-grafting. Conversely, after 3 weeks of transplantation, the primary follicular density was higher in fragments treated with Z-VAD-FMK. This improvement was associated with a decreased percentage of apoptosis in the tissue. CONCLUSIONS: In situ administration of Z-VAD-FMK slightly improves primary follicular preservation and reduces global apoptosis after 3 weeks of transplantation. Data presented herein will help to guide further researches towards a combined approach targeting multiple cell death pathways, angiogenesis stimulation, and follicular recruitment inhibition.

Supplementation of transport and freezing media with anti-apoptotic drugs improves ovarian cortex survival.

BACKGROUND: Ovarian tissue preservation is proposed to patients at risk of premature ovarian failure, but this procedure still needs to be optimized. To limit injury during ovarian tissue cryopreservation, anti-apoptotic drugs were added to the transport and freezing media of ovarian cortex tissue. METHODS: Sheep ovaries were transported, prepared and frozen in solutions containing vehicle or anti-apoptotic drugs (Z-VAD-FMK, a pan-caspase inhibitor, or sphingosine-1-phosphate (S1P), a bioactive lipid). After the tissue was thawed, the ovarian cortex was cultured for 2 or 6 days. Follicular quantification and morphological and proliferation analyses were performed on histological sections. RESULTS: After 2 days of culture, S1P improved the quality of primordial follicles; higher densities of morphologically normal and proliferative primordial follicles were found. Z-VAD-FMK displayed similar effects by preserving global primordial follicular density, but this effect was evident after 6 days of culture. This drug also improved cell proliferation after 2 and 6 days of culture. CONCLUSIONS: Our results showed that the addition of S1P or Z-VAD-FMK to the transport and freezing media prior to ovarian tissue cryopreservation improves primordial follicular quality and therefore improves global tissue survival. This should ultimately lead to improved fertility restoration after auto-transplantation.

In vitro evaluation of the anti-apoptotic drug Z-VAD-FMK on human ovarian granulosa cell lines for further use in ovarian tissue transplantation.

PURPOSE: Because ovarian granulosa cells are essential for oocyte survival, we examined three human granulosa cell lines as models to evaluate the ability of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) to prevent primordial follicle loss after ovarian tissue transplantation. METHODS: To validate the efficacy of Z-VAD-FMK, three human granulosa cell lines (GC1a, HGL5, COV434) were treated for 48 h with etoposide (50 µg/ml) and/or Z-VAD-FMK (50 µM) under normoxic conditions. To mimic the ischemic phase that occurs after ovarian fragment transplantation, cells were cultured without serum under hypoxia (1 % O(2)) and treated with Z-VAD-FMK. The metabolic activity of the cells was evaluated by WST-1 assay. Cell viability was determined by FACS analyses. The expression of apoptosis-related molecules was assessed by RT-qPCR and Western blot analyses. RESULTS: Our assessment of metabolic activity and FACS analyses in the normoxic experiments indicate that Z-VAD-FMK protects granulosa cells from etoposide-induced cell death. When cells are exposed to hypoxia and serum starvation, their metabolic activity is reduced. However, Z-VAD-FMK does not provide a protective effect. In the hypoxic experiments, the number of viable cells was not modulated, and we did not observe any modifications in the expressions of apoptosis-related molecules (p53, Bax, Bcl-xl, and poly (ADP-ribose) polymerase (PARP)). CONCLUSION: The death of granulosa cell lines was not induced in our ischemic model. Therefore, a protective effect of Z-VAD-FMK in vitro for further use in ovarian tissue transplantation could not be directly confirmed. It will be of interest to potentially use Z-VAD-FMK in vivo in xenograft models.

Isoform 165 of vascular endothelial growth factor in collagen matrix improves ovine cryopreserved ovarian tissue revascularisation after xenotransplantation in mice.

BACKGROUND: Aggressive anti-cancer treatments can result in ovarian failure. Ovarian cryopreservation has been developed to preserve the fertility of young women, but early graft revascularisation still requires improvement. METHODS: Frozen/thawed sheep ovarian cortical biopsies were embedded in collagen matrix with or without isoform 165 of vascular endothelial growth factor (VEGF165) and transplanted into ovaries of immunodeficient mice. Ovaries were chosen as transplantation sites to more closely resemble clinical conditions in which orthotopic transplantation has previously allowed several spontaneous pregnancies. RESULTS: We found that VEGF165 significantly increased the number of Dextran-FITC positive functional vessels 3 days after grafting. Dextran- fluorescein isothiocyanate (FITC) positive vessels were detectable in 53% and 29% of the mice in the VEGF-treated and control groups, respectively. Among these positive fragments, 50% in the treated group displayed mature smooth-muscle-actin-alpha (alpha-SMA) positive functional vessels compared with 0% in the control group. CD31 positive murine blood vessels were observed in 40% of the VEGF165 transplants compared with 21% of the controls. After 3 weeks, the density of murine vessels was significantly higher in the VEGF165 group. CONCLUSION: The encapsulation of ovarian tissue in collagen matrix in the presence of VEGF165 before grafting has a positive effect on functional blood vessel recruitment. It can be considered as a useful technique to be improved and further developed before human clinical applications in female cancer patients in the context of fertility preservation.

Influence of mouse strain on ovarian tissue recovery after engraftment with angiogenic factor.

BACKGROUND: For women facing gonadotoxic treatment, cryopreservation of ovarian tissue with subsequent retransplantation during remission is a promising technique for fertility preservation. However, follicle loss within grafted ovarian tissue can be caused by ischemia and progressive revascularization. Several xenograft models using different immunodeficient rodent lines are suitable for studying ovarian tissue survival and follicular viability after frozen-thawed ovarian cortex transplantation. SCID mice, which are deficient for functional B and T cells, are the most commonly used mice for ovarian xenograft studies. However, due to incomplete immunosuppression, NOD-SCID mice displaying low NK cell function and an absence of circulating complement might be more appropriate. The present study aims to define the most appropriate immunodeficient mouse strain for ovarian tissue xenotransplantation by comparing ovarian graft recovery in SCID and NOD-SCID mice following engraftment in the presence of isoform 111 of vascular endothelial growth factor. METHODS: Sheep ovarian cortex fragments were embedded in a collagen matrix, with or without VEGF111, before being stitched onto the ovaries of SCID and NOD-SCID mice. Transplants were recovered after 3 days to study early revascularization or after 3 weeks to evaluate follicle preservation and tissue fibrosis through histological analyses. RESULTS: At day 3, vessels were largely reorganized in the ovarian grafts of both mouse strains. After 3 weeks, the cortical tissue was clearly identifiable in SCID mice but not in NOD-SCID mice. Upon VEGF111 treatment, vascularization was significantly improved 3 days after transplantation in SCID mice. This increase in vessel density was correlated with better follicular preservation in SCID mice 3 weeks after transplantation. Fibrosis was not decreased by VEGF treatment in either mouse strain. CONCLUSIONS: Tissue architecture and follicular morphology were better preserved in ovarian tissues grafted in SCID mice in comparison with NOD-SCID mice. Moreover, tissue revascularization was improved in SCID mice by VEGF111 graft treatment. Thus, we consider SCID mice to be the best murine model for studying ovarian tissue xenografts.

Strategies for using the sheep ovarian cortex as a model in reproductive medicine.

OBJECTIVE: To evaluate and compare the distribution and density of primordial follicles within a whole sheep ovary and to gain insight into how to overcome the impact of natural follicular heterogeneity on the experimental results. DESIGN: Histological study. SETTING: Academic research center. ANIMALS: Five- to nine-month-old ewes. INTERVENTIONS: Freshly sampled whole sheep ovaries were collected and prepared for histological analysis. MAIN OUTCOME MEASURE(S): The follicular densities and distributions were determined for hematoxylin and eosin sections. A mathematical model was derived based on the follicle counts and Monte-Carlo simulations. RESULTS: Heterogeneous distributions and densities of primordial follicles were identified 1) for distinct areas of the same ovarian cortex, 2) between the ovaries of the same animal and 3) across different ewes. A mathematical model based on the analysis of 37,153 primordial follicles from 8 different ovaries facilitated the estimation of the number of cortical biopsies and sections that had to be analyzed to overcome such heterogeneity. CONCLUSION: The influence of physiological follicular heterogeneity on experimental and clinical results can be overcome when a definite number of cortical pieces and sections are taken into consideration.

Isoform 111 of vascular endothelial growth factor (VEGF111) improves angiogenesis of ovarian tissue xenotransplantation.

BACKGROUND: Cryopreservation of cortex ovarian tissue before anticancer therapy is a promising technique for fertility preservation mainly in children and young women. Ischemia in the early stage after ovarian graft causes massive follicle loss by apoptosis. VEGF111 is a recently described vascular endothelial growth factor (VEGF) isoform that does not bind to the extracellular matrix, diffuses extensively, and is resistant to proteolysis. These properties confer a significantly higher angiogenic potential to VEGF111 in comparison with the other VEGF isoforms. METHODS: We evaluated the morphology of cryopreserved sheep ovarian cortex grafted in the presence or absence of VEGF111. Ovarian cortex biopsies were embedded in type I collagen with or without VEGF111 addition before transplantation to severe combined immunodeficient mice ovaries. Transplants were retrieved 3 days or 3 weeks later. Follicular density, vasculature network, hemoglobin content, and cell proliferation were analyzed. RESULTS: Addition of VEGF111 increased density of functional capillaries (P=0.01) 3 days after grafting. By double immunostaining of Ki-67 and von Willebrand factor, we demonstrated that proliferating endothelial cells were found in 83% of the VEGF111 group compared with 33% in the control group (P=0.001). This angiostimulation was associated with a significant enhancement of hemoglobin content (P=0.03). Three weeks after transplantation, the number of primary follicles was significantly higher in VEGF111 grafts (P=0.02). CONCLUSION: VEGF111 accelerates blood vessel recruitment and functional angiogenesis and improves the viability of ovarian cortex by limiting ischemia and ovarian cortex damage.

Transient reduction of placental angiogenesis in PAI-1-deficient mice.

Murine placentation is associated with the invasion of maternal endometrium by trophoblasts and an extensive maternal and fetal angiogenesis. Plasminogen activator inhibitor-1 (PAI-1) is transiently produced by spongiotrophoblasts and trophoblast giant cells at 10.5-11.5 days postcoitum (dpc). Knowing the key contribution of PAI-1 in the regulation of angiogenesis, we have now analyzed the consequence of PAI-1 deficiency on murine placentation. Morphological and quantitative computer-assisted image analysis revealed abnormal placental morphology in PAI-1-/- mice at 10.5 and 12.5 dpc. At 10.5 dpc, the genetic ablation of PAI-1 resulted in a transient reduction of both maternal and fetal vascularizations in the placenta and increased trophoblast cell density. This was associated with a poorer development of the labyrinth and an extension of the decidua. A larger spongiotrophoblast layer appeared at 12.5 dpc in PAI-1-deficient mice. Placental morphology was normalized at 14.5 dpc. Microarray analyses performed on laser capture microdissected labyrinths revealed that 46 genes were differentially expressed between the two genotypes at 10.5 dpc. However, only 11 genes were still differently modulated at 14.5 dpc, when normalization of placental morphology had taken place. This transcriptomic profiling highlighted a dysregulation in the expression of placenta-related cathepsin family members. Altogether our data provide evidence for a transient impaired placental morphology in PAI-1-deficient mice that is then normalized, leading to normal embryonic development.

Differential elevation of matrix metalloproteinase expression in women exposed to levonorgestrel-releasing intrauterine system for a short or prolonged period of time.

BACKGROUND: The levonorgestrel-releasing intrauterine system (LNG-IUS) is an effective contraceptive and has many non-contraceptive health benefits. However, it is commonly associated with irregular endometrial bleeding. Metalloproteinases contribute to extracellular matrix (ECM) remodelling and regulate bleeding during the menstrual cycle. Enhanced metalloproteinase expression participates in the pathogenesis of breakthrough bleeding. Thus the objective of this study was to compare matrix metalloproteinase (MMP) expression in endometrium during luteal phase and in short-term (1 month) and long-term (> or =6 months) LNG-IUS users. METHODS: MMP expression was analysed by semi-quantitative RT-PCR and immunohistochemistry. Gelatinase activity was determined by gelatin zymography. RESULTS: MMP-1, -2, -3, -7, -9 and -12 mRNAs levels were increased, whereas that of MMP-26 was decreased in the endometrium of LNG-IUS users. MMP-1, -2, -3, -7 and -9 were localized by immunohistochemistry in all biopsies in the short-term group but in only 0-27% in the control group. The incidence of positive immunostaining for MMP-2 and -3 decreased significantly in the long-term compared with short-term LNG-IUS users. MMP-26 was localized in all biopsies from the control group but in only 14 and 25% from the short- and long-term LNG-IUS groups, respectively. In both LNG groups, the numbers of macrophages (the major source of MMP-12) was increased. CONCLUSIONS: MMP-1, active MMP-2, MMP-3, MMP-7, MMP-9 and MMP-12 are more prevalent in the short-term LNG-IUS group, suggesting their important contribution to ECM breakdown and transient bleeding. The decrease in the percentage of women expressing MMP-2 and -3 might contribute to the decreased occurrence of unwanted spotting and bleeding in long-term LNG-IUS users.

Endometrial vessel maturation in women exposed to levonorgestrel-releasing intrauterine system for a short or prolonged period of time.

BACKGROUND: Levonorgestrel-releasing intrauterine system (LNG-IUS), although inserted to reduce heavy menstruation, causes irregular early transient bleeding. The objective of the study was to document quantitative changes in endometrial vessels of short- (< or =3 months) and long-term (> or =12 months) LNG users. The area, density and maturation of endometrial vessels were quantified in 19 endometrial biopsies of women with LNG-IUS and in 10 normally ovulating patients during mid-luteal phase. METHODS: Vessel maturation was evaluated by double immunostaining using anti-von Willebrand factor (endothelial cell marker) and anti-alpha Smooth Muscle Actin (vascular smooth muscle cells) antibodies. Vessel area, number and density were quantified with a novel computer-assisted image analysis system. RESULTS: Endometrium exposed to LNG-IUS for 1-3 months displayed a 11.5-fold increase in small naked vessel number. The partially mature vessel (alphaSMA partially positive) number increased six times. After long-term LNG-IUS treatment, the immature and partially mature vessel number remained four times higher than in the control group. Vessel area and density also increased dramatically in a time-dependent pattern with LNG-IUS use. CONCLUSIONS: Levonorgestrel affects blood vessel number, area, density and maturation in a time-dependent pattern that may explain the early transient increase in breakthrough bleeding with the LNG-IUS.

Progestins regulate the expression and activity of the forkhead transcription factor FOXO1 in differentiating human endometrium.

Menstruation, or cyclic shedding of nonpregnant endometrial tissue with associated bleeding, occurs only in humans and a few other species. This breakdown of the endometrium in response to falling ovarian progesterone levels is a complex process, characterized by local leukocyte infiltration, expression and activation of matrix metalloproteinases, and apoptosis. Spontaneous decidualization (differentiation) of the stromal compartment precedes the cyclic shedding of the endometrium in various menstruating species but the mechanisms that link these processes are not understood. In this study, we identified FOXO1 as a key transcription factor responsible for mediating apoptosis of decidualized human endometrial stromal cells (HESCs) in response to progesterone withdrawal. We demonstrate that medroxyprogesterone acetate (MPA, a synthetic progestin) enhances the expression of FOXO1 in differentiating HESCs while simultaneously inducing cytoplasmic retention and inactivation of FOXO1. Withdrawal of MPA from decidualized HESCs results in rapid nuclear accumulation of FOXO1, increased BIM expression, a proapoptotic FOXO1 target gene, and cell death. Conversely, silencing of FOXO1 expression completely abolishes cell death induced by MPA withdrawal. In summary, the observation that differentiating HESCs become dependent on progesterone signaling for survival through induction and reversible inactivation of FOXO1 suggests a novel mechanism that links decidualization of the endometrium to menstruation.

Tryptophane-based biphenylsulfonamide matrix metalloproteinase inhibitors as tumor imaging agents.

AIM: As a part of our efforts to use small organic matrix metalloproteinase (MMP) inhibitors with improved characteristics for the diagnosis and treatment of different kinds of tumor tissues, biphenylsulfonamide analogues were synthesized. This study reports on the in vivo biodistribution of iodine-123-labeled biphenylsulfonide and analogues in A549 lung carcinoma inoculated into athymic mice and the evaluation of their suitability as imaging agents using a single photon emission computed tomography (SPECT) camera. METHODS: The radioiodinated carboxylic and hydroxamic MMP inhibitors 2-(4'- [(123)I]iodobiphenyl-4-sulfonylamino)-3-(1H-indol-3-yl)-propionic acid (1') and 2-(4'-[(123)I]iodobiphenyl-4- sulfonylamino)-3-(1H-indol-3-yl)-propionamide (2') were synthesized by electrophilic aromatic substitution of the tributylstannyl derivatives. Planar gamma camera imaging was performed in nu/nu athymic mice bearing an A549 tumor using a Toshiba GCA-9300A/hg SPECT camera in planar mode equipped with a high-resolution, parallel-hole collimator. RESULTS: Radiosynthesis of (1') and (2') resulted in radiochemical yields of 60 +/- 5% (n +/- 3) and 70 +/- 5% (n = 6), respectively. Evaluation of tumors induced in athymic mice by the inoculation of non-small cell lung A549 carcinoma cells, showed a tumor uptake of 0.27-0.01 percent injected dose per gram (%ID/g) (3 hours-48 hours p.i.), a tumor-blood ratio of 0.7, a tumor-muscle ratio of 1.6, and a tumor-fat ratio of 0.5 at 24 hours (p.i.) for compound 1'. For compound 2' a tumor uptake of 0.7-0.04 %ID/g (3 hours-48 hours p.i.), a postinjection tumor-blood ratio of 1.2, a tumor-muscle ratio of 3.2, and a tumor-fat ratio of 2.4 at 48 hours p.i. was observed. SPECT evaluation confirmed the results obtained from biodistribution. CONCLUSION: In vivo evaluation of these radioiodinated carboxylic and hydroxamic MMP inhibitor tracers revealed that they do not appear suitable as tumor-imaging agents.