Diana Valbuena

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.

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.

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.

Mitochondrial DNA content as a viability score in human euploid embryos: less is better.

OBJECTIVE To investigate the clinical relevance of mitochondrial DNA (mtDNA) content as a viability score in human euploid embryos. DESIGN Retrospective analysis of mtDNA content of transferred euploid embryos. SETTING Reproductive genetics laboratory. PATIENT(S) Single-embryo transfer in 270 patients who underwent preimplantation genetic screening (205 day-3 blastomere biopsies, and 65 day-5 trophectoderm biopsies), and 10 patients with double-embryo transfer (male-female). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Normalized mtDNA content versus nuclear DNA (nDNA) from transferred euploid embryos. RESULT(S) A high mtDNA copy number in euploid embryos is indicative of lower embryo viability and implantation. Using the normalized mtDNA content, we created the mitochondrial score or Mitoscore (Ms). Day-3 embryos with <34 (MsA) had an implantation rate (IR) of 59% (n = 51); those with 34-52 (MsB) had an IR of 44% (n = 52); those with 52-97 (MsC) had an IR of 42% (n = 50); and those with >97 (MsD) had an IR of 25% (n = 52). Embryos with Ms >160 (n = 22) never implanted. Day-5 embryos with <18.19 (MsA) had an IR of 81%; those with 18.19-24.15 (MsB) had an IR of 50% (n = 16); those with 24.15-50.58 (MsC) had an IR of 62% (n = 16); and those with levels >50.58 (MsD) had an IR of 18% (n = 17). Embryos with levels >60 (n = 7) never implanted. CONCLUSION(S) An increased amount of mtDNA in euploid embryos is related to poor implantation potential and may be indicative of reduced metabolic fuel during oocyte maturation. We are implementing Ms in our preimplantation genetic screening platform to prospectively analyze its clinical relevance.

Increased Adhesiveness in Cultured Endometrial-Derived Cells Is Related to the Absence of Moesin Expression

Abstract 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.

Functional Genomics of 5- to 8-Cell Stage Human Embryos by Blastomere Single-Cell cDNA Analysis

Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.

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.

The role of estrogen in uterine receptivity and blastocyst implantation

Abstract The endometrium is a specialized, hormonally regulated organ that is non adhesive for embryos throughout most of the reproductive cycle in mammals. Thus, the window of implantation is a self-limited period in which the endometrial epithelium (EE) acquires a functional and transient ovarian steroid-dependent status. The luminal EE initiates the adhesion of the developing blastocyst during this period, owing mainly to the presence of progesterone (P) after appropriate 17β-estradiol (E 2 ) priming in humans or because of the addition of E 2 after appropriate P priming in rodents. Wen-ge et al. have now demonstrated in mice that low levels of exogenous E 2 can maintain the window of receptivity for an extended period of time, whereas high doses of E 2 can rapidly initiate a refractory state. In summary, levels of E 2 within a very narrow range determine the duration of the window of implantation in uterine receptivity in mice. These outstanding results demonstrate the possibility of manipulating the receptivity window with the use of different doses of E 2 .

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

AbstractPurpose: 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 husbands 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.

Derivation and characterization of three new Spanish human embryonic stem cell lines (VAL −3 −4 −5) on human feeder and in serum-free conditions

A total of 184 human embryos, frozen for >5 years, were donated; informed consent was obtained according to Spanish law 45/2003. Survival rate was 40% and three out of 24 blastocysts (12.5%) developed into putative hESC lines, named VAL-3, VAL-4, and VAL-5. The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout DMEM supplemented with knockout serum replacement, and basic fibroblast growth factor. Fingerprinting and HLA typing of the cell lines allowed their identification and traceability. Karyotype was normal for VAL-3 (46XY), VAL-4 (46XX) and VAL-5 (46XX). All three hESC lines expressed specific markers for non-differentiation (Nanog, stage-specific embryonic antigen-4 [SSEA-4], tumour-related antigen [TRA]-1-60, and TRA-1-81) and were negative for SSEA-1. RT-PCR further demonstrated the expression of Oct-4, Sox2, Rex-1, Nanog, Cripto, Thy-1, and Lefty-A. Furthermore, they were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All three cell lines displayed high levels of telomerase activity, and were shown to successfully overcome cryopreservation and thawing. Finally, these three new hESC lines have demonstrated the potential to differentiate in vitro and in vivo (teratoma formation) into cell types originating from all three germ layers.