Expression and regulation of the tumor suppressor, SEF, during folliculogenesis in humans and mice.

Similar expression to FGF (Sef or IL17-RD), is a tumor suppressor and an inhibitor of growth factors as well as of pro-inflammatory cytokine signaling. In this study, we examined the regulation of Sef expression by gonadotropins during ovarian folliculogenesis. In sexually immature mice, in situ hybridization (ISH) localized Sef gene expression to early developing oocytes and granulosa cells (GC) but not to theca cells. Sef was also expressed in mouse ovarian endothelial cells, in the fallopian tube epithelium as well as in adipose tissue venules. SEF protein expression, determined by immunohistochemistry (IHC), correlated well with Sef mRNA expression in GC, while differential expression was noticed in oocytes. High Sef mRNA but undetectable SEF protein levels were observed in the oocytes of primary/secondary follicles, while an inverse correlation was found in the oocytes of preantral and small antral follicles. Sef mRNA expression dropped after pregnant mare's serum gonadotropin (PMSG) administration, peaked at 6-8 h after human chorionic gonadotropin (hCG) treatment, and declined by 12 h after this treatment. ISH and IHC localized the changes to oocytes and mural GC following PMSG treatment, whereas Sef expression increased in mural GC and declined in granulosa-lutein cells upon hCG treatment. The ovarian expression of SEF was confirmed using human samples. ISH localized SEF transcripts to human GC of antral follicles but not to corpora lutea. Furthermore, SEF mRNA was detected in human GC recovered from preovulatory follicles. These results are the first to demonstrate SEF expression in a healthy ovary during folliculogenesis. Hormonal regulation of its expression suggests that SEF may be an important factor involved in intra-ovarian control mechanisms.

Embryo catheter loading and embryo culture techniques: results of a worldwide Web-based survey.

PURPOSE: To identify trends in embryo catheter loading and embryo culture techniques performed worldwide. METHODS: A retrospective evaluation using the results of a web-based survey, (IVF Worldwide ( www.IVF-worldwide.com ), was performed. RESULTS: Responses from 265 centers in 71 countries were obtained. Most centers (97 %) preferred a catheter with its orifice on top, with only 3 % preferring a catheter with the orifice on its side; 41 % preferred a catheter marked for clear ultrasound view. The most commonly-reported methods of embryo loading were medium-air-embryo-air-medium (42 %), medium in catheter with embryo at end (20 %) and medium-air-embryo (15 %). In 68 % of centers the final volume of the catheter was up to 0.3 ml, with only 19 % using 0.3-0.5 ml and 1 % using 0.5-0.7 ml. Using reduced oxygen concentrations for embryo culture was divided between those who used it in combination with the two-gas system (34 %) and those who did not use it at all (39 %); 24 % reported using a three-gas system. Most clinics using reduced oxygen concentrations used it throughout the entire culture period. Half of centers (51 %) reported using reduced oxygen concentrations for the entire IVF population while 6 % reserved it only for blastocyst transfer. The use of sequential media was highly dominant with 40 % reporting its use.

Oocyte-directed depletion of connexin43 using the Cre-LoxP system leads to subfertility in female mice.

Gap junctions, predominantly comprising connexin43 (Cx43), mediate cell-to-cell communication within the ovarian follicle. However, the partaking of Cx43 in the formation of the gap junction channels, between the oocyte and the somatic cells, is controversial. We addressed this dispute by crossing females that carry a Cx43 coding region, flanked by loxP recognition sites, with males expressing the Cre recombinase under the control of Zp3 promoter. Oocytes of the resultant Zp3Cre;Gja1(lox/lox) mice did not express Cx43 and were referred to as Cx43(del/del). Unexpectedly, a decrease in Cx43 was observed in cumulus/granulosa cells of some follicles as well. Nevertheless, no histological abnormalities were detected in the ovaries of the Zp3Cre;Gja1(lox/lox) mice. Furthermore, these mice ovulated normally and developed fully functional corpora lutea. Additionally, the ovarian Cx43(del/del) oocytes were meiotically arrested and transferred Lucifer yellow to the surrounding cumulus cells. However, mating Zp3Cre;Gja1(lox/lox) females with wild-type males resulted in a reduced rate of parturition and a substantial decrease in litter size. Further examination revealed that although preimplantation development of Zp3Cre;Gja1(lox/+) embryos was normal, the blactocysts exhibited impaired implantation. Our data suggest that total ablation of Cx43 in the oocyte, combined with its decrease in the surrounding somatic cells, allows normal oogenesis and folliculogenesis, ovulation and early embryonic development but severely impairs the implantation capacity of the resulting blactocysts.

The ovarian gap junction protein connexin43: regulation by gonadotropins.

The major role of the ovarian follicle is the timely production of a mature fertilizable oocyte. This mission is accomplished by a gonadotropin-regulated, gap junction-mediated alteration between established and interrupted cell-cell communication. Recent studies have revealed that gonadotropin action on ovarian gap junctions is elicited at the transcriptional, translational and post-translational levels. Here, we review the existing information generated on the molecular mechanisms employed by the gonadotropins to elicit their effect on the ovarian gap junction protein Cx43.

Luteinizing hormone-induced connexin 43 down-regulation: inhibition of translation.

The coordinated function of the different compartments of the follicle, the oocyte and the somatic cumulus/granulosa cells, is enabled by the presence of a network of cell-to-cell communication generated by gap junctions. Connexin 43 (Cx43) is the most abundant gap junction protein expressed by the ovarian follicle. The expression of Cx43 is subjected to the control of gonadotropins as follows: FSH up-regulates, whereas LH down-regulates its levels. The aim of this study was to explore the mechanism by which LH reduces the levels of Cx43 and to identify the signal transduction pathway involved in this process. The effect of LH was studied in vitro using isolated intact ovarian follicles. The possible mediators of LH-induced Cx43 down-regulation were examined by incubating the follicles with LH in the presence or absence of inhibitors of protein kinase A (PKA) and of MAPK signaling pathways. Our experiments revealed a 3-h half-life of Cx43 in both control and LH-treated follicles, suggesting that LH did not affect the rate of Cx43 degradation. We further demonstrated that the level of Cx43 mRNA was not significantly influenced by this gonadotropin. However, upon LH administration, [(35)S]methionine incorporation into Cx43 protein was remarkably reduced. The LH-induced arrest of Cx43 synthesis was counteracted by inhibitors of both the PKA and the MAPK cascades. We show herein that LH inhibits Cx43 expression by reducing its rate of translation and that this effect is mediated by both PKA and MAPK.

Local injury to the endometrium doubles the incidence of successful pregnancies in patients undergoing in vitro fertilization.

OBJECTIVE: Exploration of the possibility that local injury of the endometrium increases the incidence of implantation. DESIGN: Prospective study. SETTING: Clinical IVF unit. PATIENT(S): A group of 134 patients, defined as good responders to hormonal stimulation, who failed to conceive during one or more cycles of IVF and embryo transfer (ET). INTERVENTION(S): The IVF treatment and ET were preceded by repeated endometrial biopsies, in a randomly selected 45 of a total of 134 patients. MAIN OUTCOME MEASURES: Outcome of IVF-ET treatments. RESULT(S): Transfer of a similar number of embryos (3.4 +/- 1.0 and 3.1 +/- 0.9 in the experimental and control patients, respectively) resulted in rates of implantation (27.7% vs. 14.2%, P =.00011), clinical pregnancy (66.7% vs. 30.3%, P =.00009), and live births per ET (48.9% vs. 22.5%, P =.016) that were more than twofold higher in the experimental group as compared to controls. CONCLUSION(S): These results suggest that IVF treatment that is preceded by endometrial biopsy doubles the chance for a take-home baby.

Implantation: mutual activity of sex steroid hormones and the immune system guarantee the maternal-embryo interaction.

Implantation is strictly dependent on the mutual interaction between a receptive endometrium and the blastocyst. Hence, synchronization between blastocyst development and the acquisition of endometrial receptivity is a prerequisite for the success of this process. This review depicts the cellular and molecular events that coordinate these complex activities. Specifically, the involvement of the sex steroid hormones, estrogen and progesterone, as well as components of the immune system, such as cytokines and specific blood cells, is elaborated.

The role of inflammation for a successful implantation.

Approximately half of all human embryo implantations result in failed pregnancy. Multiple factors may contribute to this failure, including genetic or metabolic abnormalities of the embryo. However, many of these spontaneous early abortion cases are attributed to poor uterine receptivity. Furthermore, although many fertility disorders have been overcome by a variety of assisted reproductive techniques, implantation remains the rate-limiting step for the success of the in vitro fertilization (IVF) treatments. We, as well as others, have demonstrated that endometrial biopsies performed either during the spontaneous, preceding cycle, or during the IVF cycle itself, significantly improve the rate of implantation, clinical pregnancies, and live births. These observations suggest that mechanical injury of the endometrium may enhance uterine receptivity by provoking the immune system to generate an inflammatory reaction. In strong support of this idea, we recently found that dendritic cells (DCs), an important cellular component of the innate immune system, play a critical role in successful implantation in a mouse model. In this review, we discuss the hypothesis that the injury-derived inflammation in the biopsy-treated patients generates a focus for uterine DCs and Mac accumulation that, in turn, enhance the endometrial expression of essential molecules that facilitate the interaction between the embryo and the uterine epithelium.

An in vitro model for the study of human implantation.

PROBLEM: Implantation remains the rate-limiting step for the success of in vitro fertilization. Appropriate models to study the molecular aspects of human implantation are necessary in order to improve fertility. METHODS: First trimester trophoblast cells are differentiated into blastocyst-like spheroids (BLS) by culturing them in low attachment plates. Immortalized human endometrial stromal cells and epithelial cells (ECC-1) were stably transfected with GFP or tdTomato. Co-culture experiments were monitored using Volocity imaging analysis system. RESULTS: This method demonstrates attachment and invasion of BLS, formed by trophoblast cells, into stromal cells, but not to uterine epithelial cells. CONCLUSION: We have developed an in vitro model of uterine implantation. The manipulation of this system allows for dual color monitoring of the cells over time. Additionally, specific compounds can be added to the culture media to test how this may affect implantation and invasion. This model is a helpful tool in understanding the complexity of human implantation.

Inflammation and implantation.

Approximately half of all human embryo implantations result in failed pregnancy. Multiple factors may contribute to this failure, including genetic or metabolic abnormalities of the embryo. However, many of these spontaneous early abortion cases are attributed to poor uterine receptivity. Furthermore, although many fertility disorders have been overcome by a variety of assisted reproductive techniques, implantation remains the rate-limiting step for the success of the in vitro fertilization (IVF) treatments. It has been demonstrated that endometrial biopsies performed either during the spontaneous, preceding cycle, or during the IVF cycle itself, significantly improve the rate of implantation, clinical pregnancies and live births. These observations suggest that mechanical injury of the endometrium may enhance uterine receptivity by provoking the immune system to generate an inflammatory reaction. In strong support of this idea, we recently found that dendritic cells (DCs), an important cellular component of the innate immune system, play a critical role in successful implantation in a mouse model. In this review, we discuss the hypothesis that the injury-derived inflammation in the biopsy-treated patients generates a focus for uterine DCs accumulation that, in turn, enhances the endometrial expression of essential molecules, which facilitate the interaction between the embryo and the uterine epithelium.

Local injury of the endometrium induces an inflammatory response that promotes successful implantation.

OBJECTIVE: To study whether an injury-induced inflammation might be the mechanism underlying the favorable effect of endometrial biopsy on the implantation rate in in vitro fertilization (IVF) patients. DESIGN: Controlled clinical study. SETTING: A medical center IVF unit and a research institute. PATIENT(S): Women undergoing IVF who had previous failed treatment cycles. INTERVENTION(S): Endometrial samples were collected from two groups of patients on day 21 of their spontaneous menstrual cycle. The experimental, but not the control group underwent prior biopsy treatment on days 8 or/and 11 to 13 of that same cycle. MAIN OUTCOME MEASURE(S): Abundance of immune cells, cytokines/chemokines level, correlation between these parameters and pregnancy outcome. RESULT(S): A statistically significantly higher amount of macrophages/dendritic cells (HLA-DR+ CD11c+ cells) and elevated proinflammatory cytokines, tumor necrosis factor-α (TNF-α), growth-regulated oncogene-α (GRO-α), interleukin-15 (IL-15), and macrophage inflammatory protein 1B (MIP-1B), were detected in day-21 endometrial samples of the experimental group. A direct stimulatory effect of TNF-α on MIP-1B, GRO-α, and IL-15 messenger RNA (mRNA) expression was demonstrated. A positive correlation was found between the levels of macrophages/dendritic cells, MIP-1B expression, and TNF-α expression and the pregnancy outcome. CONCLUSION(S): A biopsy-induced inflammatory response may facilitate the preparation of the endometrium for implantation. Increased MIP-1B expression could possibly serve for prediction of implantation competence.

Successful pregnancy and delivery of a healthy baby after endometrial biopsy treatment in an in vitro fertilization patient with severe Asherman syndrome.

OBJECTIVE: To implement the procedure of endometrial biopsy in a case of severe Asherman syndrome as a possible treatment to increase uterine receptivity. DESIGN: Case report. SETTING: IVF Unit, Kaplan Medical Center, Rehovot, Israel. PATIENT(S): A 29-year-old patient with severe Asherman syndrome, who underwent six operative hysteroscopies combined with hormonal treatment, and no functional receptive endometrium was achieved. INTERVENTION(S): We performed three endometrial biopsies on days 8, 12, and 21 of a progyluton-induced menstrual cycle, and a fourth biopsy on day 21 of the next induced menstrual cycle. After that cycle the patient underwent an IVF treatment. MAIN OUTCOME MEASURE(S): Ultrasound measurement of endometrial thickness, serum beta-hCG, sonography test for the presence of a gestational sac with heartbeat, and pregnancy follow-up until birth. RESULT(S): Biopsy treatment increased the thickness of the endometrium from unobservable by sonography to 7 mm on the day of hCG administration. The next IVF cycle resulted in implantation of an embryo and the birth of a healthy baby boy. CONCLUSION(S): Repeated endometrial biopsies may be used in patients with Asherman syndrome immediately after forming a uterine cavity by hysteroscopy to improve its receptivity.

Endometrial biopsy-induced gene modulation: first evidence for the expression of bladder-transmembranal uroplakin Ib in human endometrium.

OBJECTIVE: To explore the possibility that endometrial injury modulates the expression of specific genes that may increase uterine receptivity. DESIGN: Controlled clinical study. SETTING: Clinical IVF unit and academic research center. PATIENT(S): IVF patients with 28- to 30-day menstrual cycles. INTERVENTION(S): Endometrial biopsies from two groups of patients were collected on days 20-21 of their spontaneous menstrual cycle. The experimental, but not the control, group underwent biopsies on days 11-13 and 21-24 of their preceding cycle. MAIN OUTCOME MEASURE(S): Global endometrial gene expression and specific analysis of uroplakin Ib (UPIb) mRNA level throughout the menstrual cycle. RESULT(S): Local injury modulated the expression of a wide variety of genes. One of the prominently up-regulated genes was the bladder transmembranal protein, UPIb, whose expression by the endometrium is shown here for the first time. Endometrial UPIb mRNA increases after biopsy in the same cycle wct 2with an additional elevation in the following cycle. Immunohistochemical analysis localized the UPIb protein to the glandular-epithelial cells. Genes encoding other membrane proteins such as adipose differentiation-related protein and mucin 1, transmembrane, were also up-regulated. CONCLUSION(S): The biopsy-induced increase in the expression of UPIb and other genes encoding membrane proteins supports the possible importance of the membrane structure and stability during implantation. The specific role of UPIb in uterine receptivity should be elucidated.

Connexin43 in rat oocytes: developmental modulation of its phosphorylation.

It is well established that the 43-kDa connexin (Cx43) is predominantly expressed by ovarian somatic cells, whereas the identity of the connexins contributed by the oocyte to form gap junctions with its neighboring cells is not fully elucidated. Our study aimed to examine oocytes for the expression and regulation of Cx43 throughout oogenesis. Growing and fully grown rat oocytes that were meiotically incompetent and competent, respectively, were examined. Fully grown oocytes were analyzed either before or after reinitiation of meiosis as well as at the second meiotic metaphase. Immunofluorescent analysis of zona pellucida-free oocytes using conventional and confocal microscopy demonstrated a characteristic pattern of punctuated staining of Cx43 on the oolema. Immunogold electron microscopy localized Cx43 to the oocyte surface and the microvillar processes. Reverse transcriptase-polymerase chain reaction and Western blot analysis revealed similar amounts of Cx43 gene and protein in oocytes of different developmental stages. However, a relative increase in the phosphorylated forms of the protein was observed in fully grown oocytes that had completed their maturation. Our findings demonstrate that rat oocytes express a developmentally regulated Cx43. They further suggest that homotypic gap junctions that consist of Cx43 may be present between rat oocytes and their adjacent cumulus cells.