Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.

STUDY QUESTION: What is the origin and composition of cell-free DNA in human embryo spent culture media? SUMMARY ANSWER: Cell-free DNA from human embryo spent culture media represents a mix of maternal and embryonic DNA, and the mixture can be more complex for mosaic embryos. WHAT IS KNOWN ALREADY: In 2016, ~300 000 human embryos were chromosomally and/or genetically analyzed using preimplantation genetic testing for aneuploidies (PGT-A) or monogenic disorders (PGT-M) before transfer into the uterus. While progress in genetic techniques has enabled analysis of the full karyotype in a single cell with high sensitivity and specificity, these approaches still require an embryo biopsy. Thus, non-invasive techniques are sought as an alternative. STUDY DESIGN, SIZE, DURATION: This study was based on a total of 113 human embryos undergoing trophectoderm biopsy as part of PGT-A analysis. For each embryo, the spent culture media used between Day 3 and Day 5 of development were collected for cell-free DNA analysis. In addition to the 113 spent culture media samples, 28 media drops without embryo contact were cultured in parallel under the same conditions to use as controls. In total, 141 media samples were collected and divided into two groups: one for direct DNA quantification (53 spent culture media and 17 controls), the other for whole-genome amplification (60 spent culture media and 11 controls) and subsequent quantification. Some samples with amplified DNA (N = 56) were used for aneuploidy testing by next-generation sequencing; of those, 35 samples underwent single-nucleotide polymorphism (SNP) sequencing to detect maternal contamination. Finally, from the 35 spent culture media analyzed by SNP sequencing, 12 whole blastocysts were analyzed by fluorescence in situ hybridization (FISH) to determine the level of mosaicism in each embryo, as a possible origin for discordance between sample types. PARTICIPANTS/MATERIALS, SETTING, METHODS: Trophectoderm biopsies and culture media samples (20 µl) underwent whole-genome amplification, then libraries were generated and sequenced for an aneuploidy study. For SNP sequencing, triads including trophectoderm DNA, cell-free DNA, and follicular fluid DNA were analyzed. In total, 124 SNPs were included with 90 SNPs distributed among all autosomes and 34 SNPs located on chromosome Y. Finally, 12 whole blastocysts were fixed and individual cells were analyzed by FISH using telomeric/centromeric probes for the affected chromosomes. MAIN RESULTS AND THE ROLE OF CHANCE: We found a higher quantity of cell-free DNA in spent culture media co-cultured with embryos versus control media samples (P ≤ 0.001). The presence of cell-free DNA in the spent culture media enabled a chromosomal diagnosis, although results differed from those of trophectoderm biopsy analysis in most cases (67%). Discordant results were mainly attributable to a high percentage of maternal DNA in the spent culture media, with a median percentage of embryonic DNA estimated at 8%. Finally, from the discordant cases, 91.7% of whole blastocysts analyzed by FISH were mosaic and 75% of the analyzed chromosomes were concordant with the trophectoderm DNA diagnosis instead of the cell-free DNA result. LIMITATIONS, REASONS FOR CAUTION: This study was limited by the sample size and the number of cells analyzed by FISH. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to combine chromosomal analysis of cell-free DNA, SNP sequencing to identify maternal contamination, and whole-blastocyst analysis for detecting mosaicism. Our results provide a better understanding of the origin of cell-free DNA in spent culture media, offering an important step toward developing future non-invasive karyotyping that must rely on the specific identification of DNA released from human embryos. STUDY FUNDING/ COMPETING INTEREST: This work was funded by Igenomix S.L. There are no competing interests.

Advances in the systemic treatment of therapeutic approaches in biliary tract cancer.

INTRODUCTION: Biliary tract cancers (BTCs) are a rare and heterogenous group with an increasing incidence and high mortality rate. The estimated new cases and deaths of BTC worldwide are increasing, but the incidence and mortality rates in South East Asia are the highest worldwide, representing a real public health problem in these regions. BTC has a poor prognosis with a median overall survival <12 months. Thus, an urgent unmet clinical need for BTC patients exists and must be addressed. RESULTS: The backbone treatment of these malignancies is chemotherapy in first- and second-line setting, but in the last decade a rich molecular landscape has been discovered, expanding conceivable treatment options. Some druggable molecular aberrations can be treated with new targeted therapies and have already demonstrated efficacy in patients with BTC, improving clinical outcomes, such as the FGFR2 or IDH1 inhibitors. Many other molecular alterations are being discovered and the treatment of BTC will change in the near future from our current clinical practice. CONCLUSIONS: In this review we discuss the epidemiology, molecular characteristics, present treatment approaches, review the recent therapeutic advances, and explore future directions for patients with BTC. Due to the rich molecular landscape of BTC, molecular profiling should be carried out early. Ongoing research will bring new targeted treatments and immunotherapy in the near future.

New techniques on embryo manipulation.

For many years, experience has been accumulated on embryo and gamete manipulation in livestock animals. The present work is a review of these techniques and their possible application in human embryology in specific cases. It is possible to manipulate gametes at different levels, producing paternal or maternal haploid embryos (hemicloning), using different techniques including nuclear transfer. At the embryonic stage, considering practical, ethical and legal issues, techniques will be reviewed that include cloning and embryo splitting at the cleavage stage, morula, or blastocyst stage.

Lower implantation rates in high responders: evidence for an altered endocrine milieu during the preimplantation period.

OBJECTIVE: To determine serum E2 and P levels around the time of implantation in normal and high IVF responders. SETTING: In Vitro Fertilization program at the Instituto Valenciano de Infertilidad. PATIENTS: Twenty-nine women undergoing IVF, who accepted to be studied daily, were classified according to the number of oocytes retrieved in normal (n = 16) and high responders (n = 13). DESIGN: Prospective study in which blood was drawn daily from the day of hCG administration (day 0) up to 7 days later (day 6). MAIN OUTCOME MEASUREMENTS: In vitro fertilization parameters (number of ampules, FSH-hMG, number of oocytes, fertilization rates, number of transferred embryos, implantation rates, and pregnancy rates); serum E2 and P levels during the 7 days of the study. RESULTS: Implantation rate was significantly higher in normal (18.5%) as compared with high (0%) responders. Estradiol and P levels were elevated significantly in high responders. The E2:P ratio was significantly different between normal and high responders during the preimplantation period. Pregnancy and implantation rates decreased as serum E2 levels increased on days 4 to 6 of the study. CONCLUSIONS: A different endocrine milieu between normal and high responders is detected by daily steroid measurements up to the preimplantation period, suggesting that this difference could be responsible for an impaired implantation in high responder patients undergoing IVF. An increase in serum E2 levels seems to be the cause of this difference.

The follicular endocrine environment in stimulated cycles of women with endometriosis: steroid levels and embryo quality.

OBJECTIVE: To assess the endocrine milieu in follicles of stimulated cycles comparing women with and without endometriosis. Steroids were measured in follicular fluid (FF) and in in vitro culture of granulosa-luteal cells, and this status was related to the quality of the embryos obtained after IVF. DESIGN: Case-control study. SETTING: IVF program at the Instituto Valenciano de Infertilidad. PATIENT(S): Twenty-four women with laparoscopically documented endometriosis and 26 controls undergoing IVF. INTERVENTION(S): Individual follicular aspiration, oocyte isolation, FF storage, and preparation of luteinized granulosa cells for culture; oocyte insemination and embryo cleavage in standard IVF. MAIN OUTCOME MEASURE(S): Serum (day of ovum pickup) and FF measurements of estradiol, progesterone, testosterone, and androstenedione. Secretion of progesterone was measured in the cell-conditioned medium. Results were compared between patients with endometriosis and controls, as well as between oocytes that yielded embryos of different quality. RESULT(S): Levels of progesterone in the FF increased with the severity of the disease, whereas testosterone accumulation in the FF decreased with the severity of the disease. An increase in progesterone accumulation in vitro was observed in basal and hCG-induced granulosa cell cultures. No difference was observed in terms of embryo quality, and no steroid marker was able to identify follicles with oocytes that displayed embryos of good or bad quality under the inverted microscope. CONCLUSION(S): The data show differences in the steroidogenesis of follicles from stimulated women with and without endometriosis. These changes indicate good endocrine health but are not predictive of embryo quality.

Age does not affect uterine resistance to vascular flow in patients undergoing oocyte donation.

OBJECTIVE: To determine whether uterine vasculature is affected by age using oocyte donation as an in vivo model. DESIGN: Prospective longitudinal study in which recipients were grouped according to age. They underwent a successful oocyte donation cycle, and single pregnancies were followed during the first trimester by color Doppler ultrasound in uterine arteries. SETTING: Oocyte donation and IVF program at the Instituto Valenciano de Infertilidad. INTERVENTIONS: Serum E2, P, and hCG levels in single ovum donation pregnancies; pulsatility and resistance indexes in uterine arteries during initial pregnancy. RESULTS: Similar serum levels of E2, P, and hCG in both groups of patients were observed. There was no difference between groups regarding the flow indexes analyzed. CONCLUSIONS: The increased incidence of early pregnancy losses observed in patients > 40 years cannot be attributed to defective response of uterine vasculature to exogenous hormone replacement. Thus, uterine aging does not appear to be a factor influencing the poor reproductive performance of women with advancing age.

Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo.

OBJECTIVE: To investigate whether the deleterious effect of E(2) on embryonic implantation is due to a direct effect on the endometrium, on the embryo, or both. DESIGN: Prospective, controlled in vitro study. SETTING: Tertiary infertility center. PATIENT(S): Fertile patients in the luteal phase with histologically normal endometrium who were attending the infertility clinic as oocyte donors (n = 14). INTERVENTION(S): E(2) dose-response (0, 10(-8), 10(-7), 10(-6), 10(-5), and 10(-4) M) and time course (day 2 vs. day 5) experiments were performed in an in vitro embryo adhesion assay composed of human polarized endometrial epithelial cells obtained from fertile patients and mouse embryos. MAIN OUTCOME MEASURE(S): Blastocyst formation rate and embryo adhesion rate. RESULTS: Monolayers of polarized endometrial epithelial cells expressed ERalpha at the mRNA level. The E(2) dose response of blastocysts with polarized endometrial epithelial cells (n = 235) demonstrated a progressive reduction in embryonic adhesion that was statistically significant at 10(-6) M. When polarized endometrial epithelial cells were treated alone with increasing doses of E(2) for 3 days and E(2) was then removed and blastocysts added (n = 410), embryonic adhesion was not significantly reduced, except at 10(-4) M. When 2-day mouse embryos (n = 609) were treated with increasing E(2) concentrations until day 5, the rate of blastocyst formation significantly decreased at a concentration >or= 10(-6) M, and embryonic adhesion decreased when blastocysts (n = 400) were obtained at a concentration >or= 10(-7) M. Time course experiments of embryos cultured for 2 days with polarized endometrial epithelial cells (n = 426) showed that the adhesion rate was higher at E(2) levels of 10(-7), 10(-6) and 10(-5) M compared with embryos cultured for 5 days (n = 495). CONCLUSION(S): High E(2) levels are deleterious to embryo adhesion in vitro, mainly because they have a direct toxic effect on the embryo that may occur at the cleavage stage.

Interleukin-1 receptor antagonist prevents embryonic implantation by a direct effect on the endometrial epithelium.

OBJECTIVE: To investigate the embryonic and/or endometrial molecular mechanisms underlying the antiimplantation effect of interleukin-1 receptor antagonist (IL-1ra). DESIGN: Controlled experiment. SETTING: Animal facilities at Stanford University and laboratories of the Instituto Valenciano de Infertilidad and the University of Sydney. ANIMAL(S): Twelve-week-old B6C3F-1 female mice. INTERVENTION(S): Intraperitoneal injections of recombinant human IL-1ra during the periimplantation period. MAIN OUTCOME MEASURE(S): Implantation sites, embryonic morphology, and viability. Polymerase chain reaction and immunohistochemistry for integrins and extracellular matrices and transmission electron microscopy of endometrium in IL-1ra-treated versus control animals. RESULT(S): Pregnancy rates in control and IL-1ra-injected animals were 60% and 13%, respectively. At day 8 of pregnancy, flushing of uteri obtained from the treated group resulted in 32 blastocysts. Six pseudopregnant animals received IL-1ra-treated blastocysts (left horn) and control blastocysts (right horn), resulting in one pregnancy, with two embryos and one embryo in the left and right horns, respectively. At day 4 of pregnancy, IL- 1ra down-regulated alpha4 mRNA with use of the polymerase chain reaction. Immunohistochemistry showed a decrease of alpha4, alpha v, and beta3, and transmission electron microscopy revealed inhibition of transformation of the plasma membrane. CONCLUSION(S): Impairment of embryonic adhesion with IL-1ra is mediated through a direct effect on transformation of the epithelial plasma membrane at the time of implantation as a result of down-regulation of alpha4, alpha v, and beta3.

Increasing uterine receptivity by decreasing estradiol levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen.

OBJECTIVE: To analyze the effect on uterine receptivity of a decrease in E2 levels during the preimplantation period with the use of a step-down regimen in high responders undergoing IVF. DESIGN: Prospective controlled clinical study. SETTING: The Instituto Valenciano de Infertilidad. PATIENT(S): High responders in whom at least one previous IVF attempt failed in which 3-4 good-quality embryos were transferred and E2 levels were >3,000 pg/mL on the day of hCG administration. INTERVENTION(S): Gonadotropins were administered according to two different protocols. Blood samples were collected and IVF was performed. MAIN OUTCOME MEASURE(S): Serum E2 levels and reproductive outcome of IVF. RESULT(S): Estradiol levels on the day of hCG administration and throughout the preimplantation period and the number of oocytes collected were significantly lower with the use of the step-down regimen than during the previous failed cycle in which the standard protocol was used. The fertilization rate was similar and the number of good-quality embryos transferred was comparable. However, the implantation and pregnancy rates were significantly improved in patients who underwent the step-down regimen compared with those who received the standard protocol. CONCLUSION(S): With the use of a step-down regimen with FSH in high responders, our clinical results demonstrate that uterine receptivity can be improved when E2 levels are decreased during the preimplantation period.

Embryonic implantation.

Embryo implantation is a progressive process which requires a communication between two different organisms, and consists of three consecutive phases: apposition, adhesion and invasion. It must be realized throughout the period called "window of implantation", which is characterized by morphological and biochemical changes in the endometrium, as the plasma membrane transformation and the presence of some specific adhesion molecules, chemokines, cytokines, growth factors, and invasive proteinases. All of then acting in a paracrine/autocrine manner, and drive by endocrine hormones.

Embryonic regulation of endometrial molecules in human implantation.

Embryonic implantation requires co-ordinated development of the blastocyst and the maternal endometrium. Considerable advances have been made in understanding the cell biology of these partner tissues separately. Nevertheless, communication between these tissues and the reciprocal effects of these tissues on each other constitutes an exciting and as yet unsolved paradigm in reproductive medicine. Crosstalk between the embryo, endometrium and the corpus luteum is known to occur in ruminants and primates, and, more specifically, endometrial-embryonic interactions are reported in rodents and primates. In this review, an in vitro model for analysing the interactions between human endometrial epithelium and blastocyst in the adhesion phase of embryonic implantation is presented. The results of investigations of embryonic regulation of endometrial epithelial molecules such as adhesion molecules, mucins, chemokines and cytoskeleton proteins are also presented.

Clinical evidence for a detrimental effect on uterine receptivity of high serum oestradiol concentrations in high and normal responder patients.

This study was undertaken to investigate an empirical observation that 'high responder patients have poorer in-vitro fertilization (IVF) outcome than normal responder patients'. The aim of our study was to analyse the effect of high serum oestradiol and progesterone concentrations at the day of human chorionic gonadotrophin (HCG) administration on endometrial receptivity and oocyte-embryo quality in high and normal responder patients. The IVF patients were divided into two groups: 59 high responder patients who voluntarily donated some of their oocytes, and a control group consisting of 105 normal responder patients. Both groups were compared in terms of the number and quality of oocytes retrieved, embryos transferred, fertilization, implantation and gestation rates, serum oestradiol and progesterone concentrations and the oestradiol:progesterone ratio on the day of HCG injection. To ascertain oocyte-embryo quality, a second control group of 96 women undergoing oocyte donation (receiving oocytes from high responder patients) was considered. To assess the impact of steroid concentrations on endometrial receptivity, high responder patients were divided into two subgroups according to oestradiol concentration, above or below the minimal oestradiol and progesterone concentrations (mean--SD) in this group. The normal responder patients were divided into two subgroups according to oestradiol concentration, above or below the maximal oestradiol and progesterone concentrations (mean+SD) in this group. To assess further the relevance of oestradiol concentration on endometrial receptivity, patients were divided into different subgroups according to increasing oestradiol concentration, regardless of whether they were high or normal responders. High responder patients had significantly decreased implantation and pregnancy rates per cycle compared with normal responder patients (33.3 versus 16.3 and 11.1 versus 5.4% respectively; P < 0.05). The results of 108 embryo transfers in 91 recipients who received oocytes from the high responder group showed normal embryo quality. Implantation rates and pregnancies per cycle were significantly lower in high responder patients with serum oestradiol concentrations > 1700 pg/ml compared with those having oestradiol concentrations < or = 1700 pg/ml, as well as in normal responder patients with serum oestradiol concentrations > 2200 pg/ml compared with those having oestradiol concentrations < or = 2200 pg/ml. Considering all the patients together, significant decreases in pregnancy and implantation rates were observed when oestradiol concentrations were > 2500 pg/ml compared with patients having lower oestradiol concentrations. Our clinical results demonstrate that high serum oestradiol concentrations on the day of HCG injection in high and normal responder patients, regardless of the number of oocytes retrieved and the serum progesterone concentration, are detrimental to uterine receptivity without affecting embryo quality.

Interleukin 1 beta regulates Vero cell interleukin-1 receptor type I messenger ribonucleic acid expression.

Embryos cocultured with Vero cells display enhanced development in vitro. This could be due to an interaction between the embryo and cellular layer mediated by paracrine cytokines. The interleukin-1 (IL-1) system is composed of two IL-1 agonists, IL-1 receptor antagonist (IL-1ra), and two major IL-1 receptors (IL-1R tII). In this study, we measured Vero cell expression of IL-1 system mRNAs with reverse transcription polymerase chain reaction (RT-PCR) and validated the results with an immunohistochemistry study. RT-PCR revealed beta-actin and IL-1R tI mRNA expression in Vero cell cocultures without detectable IL-1 beta and IL-1ra mRNA expression. To determine the effect of IL-1 beta on IL-1R tI mRNA expression in Vero cells, quantitative competitive PCR methodology was developed. A competitive cDNA fragment was coamplified as an internal standard with the target cDNA sequence of IL-1R tI, showing a 50% decrease in Vero cell IL-1R tI cDNA cultured in the presence of IL-1 beta (100 IU/mI) compared to control Vero cell cultures (62.5 fg vs. 125 fg). Down-regulation of IL-1R tI mRNA by IL-1 beta is dose-dependent, with increasing concentrations (0-1000) IU/ml) of IL-1 beta producing progressive attenuation of IL-1R tI expression. Treatment with anti-IL-1 beta antibody ablate the inhibitory effect of IL-1 beta (100 IU/ml) on IL-1R tI mRNA expression. Immunostaining confirmed the presence of IL-1r tI protein in Vero cells. These results demonstrate the presence of IL-1 R tI in Vero cell monolayers and regulation of Il-1R tI mRNA by IL-1 beta.

Factors responsible for multiple pregnancies after ovarian stimulation and intrauterine insemination with gonadotropins.

PURPOSE: The present study was undertaken in order to analyze possible factors that could be responsible for multiple pregnancies in normoovulatory women undergoing superovulation with gonadotropins and intrauterine artificial insemination. METHODS: We retrospectively analyzed several clinical parameters in patients that achieved gestation with this treatment. Patients were divided into two groups depending on sperm origin (husband and donor sperm). Furthermore, they were subclassified as follows: (a) cycles resulting in single pregnancies (n = 366), (b) cycles ending in multiple pregnancies (n = 126), and (c) a control group composed of unsuccessful cycles (n = 366). RESULTS: In cycles employing husband's sperm, the age, number of cycles necessary to reach pregnancy, serum estradiol (E2) levels, and number of follicles were significantly (P < 0.05) different in multiple pregnancies compared to single or nonpregnant cycles. In donor insemination, women with multiple pregnancies were significantly younger than nonpregnant patients. There was a significant increase in the number of follicles developed (P < 0.00001) and serum E2 levels on the day of hCG (P < 0.05) in multiple compared to single pregnancies and unsuccessful cycles. The number of motile sperm in the insemination specimen was not different among the established groups. When both types of treatments were grouped, pregnant patients were significantly (P < 0.00001) younger than women with failed cycles. In addition, multifetal pregnancies were significantly (P < 0.05) more frequent in women < 30 years old. E2 production was significantly (P < 0.00008) higher in twin and multifetal pregnancies than in single or nonpregnant cycles. Follicular development was also significantly (P < 0.00001) higher in twin and multifetal pregnancies compared to failed cycles. CONCLUSIONS: The results suggest that young women (< 30 years) who develop more than six follicles with E2 > 1000 pg/ml when stimulated with gonadotropins are at higher risk of multiple gestation. These data may be helpful in preventing this undesired complication of assisted reproduction techniques.

Effect of disruption versus continuation of gonadotrophin-releasing agonist after human chorionic gonadotrophin administration on corpus luteum function in patients undergoing ovulation induction for in-vitro fertilization.

Previous studies have described the luteolytic effect of gonadotrophin-releasing hormone agonist (GnRHa) administered in the early luteal phase. The present work was undertaken to compare in a prospective and randomized design the effect of disruption versus continuation of daily GnRHa after human chorionic gonadotrophin (HCG) administration on corpus luteum function in patients undergoing ovulation induction for in-vitro fertilization (IVF). Two different studies were designed and a total of 38 ovum donors, aged 23-30 years, were included. In the first study, the effect of GnRHa on the early luteal phase of IVF-stimulated cycles was investigated (n = 27); the patients were divided into two groups, according to whether they stopped (n = 13) or continued with daily GnRHa injections (n = 14) for an additional period of 15 days after HCG administration. Blood was drawn from luteal phase days 2 to 6 (day 0 = day of HCG administration) and oestradiol and progesterone concentrations were analysed. The second study focused on the effects of continuation versus disruption of GnRHa administration in the mid-late luteal phase. A similar design was employed including six patients who stopped GnRH on day 0 and five other women who continued GnRHa for 15 days after HCG administration. In this second study, blood was drawn from days 5 to 11 and oestradiol, progesterone and luteinizing hormone (LH) concentrations were analysed. IVF parameters were similar in both groups. The results indicate that continuous GnRHa administration, after HCG injection, does not produce changes in oestradiol, progesterone and LH concentrations in the early, mid- and late luteal phases compared to those patients in whom GnRHa is discontinued at the day of HCG administration. The present work demonstrates that, when ovulation induction is performed, the corpus luteum is driven primarily by the HCG, regardless of the administration or disruption of GnRHa in the luteal phase. This suggests that the lack of differences between continuation versus disruption of GnRHa may be due to the accumulation of the product over the previous 2-3 weeks of treatment.

Ovarian stimulation and endometrial receptivity.

Endometrial receptivity is a limiting step in the success of in-vitro fertilization (IVF). To investigate this issue, we selected a specific population of high responder patients in whom implantation was impaired, even when good quality embryos were transferred. We present a series of studies showing that high oestradiol concentrations on the day of human chorionic gonadotrophin (HCG) administration are detrimental to uterine receptivity. In addition, we suggest clinical strategies to improve endometrial receptivity in high responder patients using a step-down regimen.

Embryonic regulation in implantation.

Maternal steroid hormones play a critical role in establishing the receptive phase of implantation. In addition to that, the embryo is able to modulate endometrial molecules during the apposition phase (chemokines) and the adhesion phase (adhesion and anti-adhesion molecules). Moreover, the human embryo also exerts a coordinated regulation of endometrial epithelial apoptosis during these implantation phases. In this work, we analyze the embryonic regulation of implantation in humans using an in vitro model.

The human blastocyst regulates endometrial epithelial apoptosis in embryonic adhesion.

The implanting blastocyst must appose and adhere to the endometrial epithelium and, subsequently, invade it. Locally regulated uterine epithelial apoptosis induced by the embryo is a crucial step of the epithelial invasion in rodents. To address the physiological relevance of this process in humans, we investigated the effect of single human blastocysts on the regulation of apoptosis in cultured human endometrial epithelial cells (hEEC) in both apposition and adhesion phases of implantation. Here, we report a co-ordinated embryonic regulation of hEEC apoptosis. In the apposition phase, the presence of a blastocyst rescues hEEC from the apoptotic pathway. However, when the human blastocyst adheres to the hEEC monolayer, it induces a paracrine apoptotic reaction. Fas ligand (Fas-L) was present at the embryonic trophoectoderm. Fas was localized at the apical cell surface of hEEC, and flow cytometry revealed that 60% of hEEC express Fas. Neutralizing adhesion assays revealed that the Fas/Fas-L death system may be an important mechanism to cross the epithelial barrier, which is crucial for embryonic adhesion, and the manipulation of this system could have potential clinical implications as an interceptive mechanism.

Oocyte donation in women with recurrent pregnancy loss.

The prognosis of couples with recurrent miscarriage is controversial despite efforts made during this century to learn about the physiopathology and treatment of this troublesome condition. Here we present our experiences of employing oocyte donation in eight couples in whom the woman was a low responder to gonadotrophin stimulation and had a previous history of recurrent abortion with negative routine infertility work-up for repeated pregnancy loss. Patients were desensitized with gonadotrophin-releasing hormone analogues and supplemented with oestradiol valerate for a minimum of 15 days until oocytes were donated from in-vitro fertilization and fertile donors. Then, progesterone was added until day 100 of pregnancy. A total of 12 oocyte donation cycles were performed in these patients. Clinical pregnancy and delivery rates per cycle were 75.0 and 66.6% respectively. The delivery rate per patient was 85.7% in this series, and the miscarriage rate per cycle was 11.1%. The results of ovum donation compared favourably with low responders without a history of recurrent abortion undergoing this treatment during the study period. These results strongly suggest that the oocyte may be the origin of infertility in women with idiopathic recurrent miscarriages. In addition, the results question the role of maternal local and systemic factors in early recurrent pregnancy loss, as well as the paternal contribution to its aetiology.

Increased adhesiveness in cultured endometrial-derived cells is related to the absence of moesin expression.

Human endometrial epithelial cells (EECs) are nonadhesive for embryos throughout most of the menstrual cycle. During the so-called implantation window, the apical plasma membrane of EECs acquire adhesive properties by undergoing a series of morphological and biochemical changes. The human endometrial-derived epithelial cell line, RL95-2, serves as an in vitro model for receptive uterine epithelium because of its high adhesiveness for trophoblast-derived cells. In contrast, the HEC-1-A cell line, which displays poor adhesive properties for trophoblast cells, is considered to be less receptive. The ezrin, radixin, and moesin protein family members, which are present underneath the apical plasma membrane, potentially act to link the cytoskeleton and membrane proteins. In the present study, we have further investigated the adhesive features in these two unrelated endometrial-derived cell lines using an established in vitro model for embryonic adhesion. We have also analyzed the protein pattern and mRNA expression of ezrin and moesin in RL95-2 cells versus HEC-1-A cells. The results demonstrate that RL95-2 cells were indeed more receptive (81% blastocyst adhesion) compared with HEC-1-A cells (46% blastocyst adhesion). An intermediate adhesion rate was found in primary EECs cultured on extracellular matrix gel, thus allowing a partial polarization of these cells (67% blastocyst adhesion). Furthermore, we found that moesin was absent from RL95-2 cells. In contrast, ezrin is expressed in both cell lines, yet it is reduced in adherent RL95-2 cells. Data are in agreement with the hypothesis that uterine receptivity requires down-regulation or absence of moesin, which is a less-polarized actin cytoskeleton.