María José de los Santos

Effects of aging on the human ovary: the secretion of immunoreactive α-inhibin and progesterone

Objective To investigate the changes induced by age in the function and secretory pattern of the human ovary. Immunoreactive α -inhibin, E 2 , and P secretion in vivo and in vitro have been compared in two different populations. Design Prospective study. Women undergoing IVF-ET were divided into two groups according to age: group 1 (32.0±0.7years; mean±SEM) and group 2 (40.3±0.3years). Setting In vitro fertilization program at the Instituto Valenciano de Infertilidad. Patients A total of 33 infertile women with regular menses, undergoing IVF-ET. Interventions Follicle aspiration performed by transvaginal ultrasound. Four follicles per patient were aspirated in individual plastic tubes. Granulosa-luteal cells isolated with Percoll columns and cultured in vitro up to 4days in the presence of hCG. Main Outcome Measures In vitro fertilization parameters, serum levels of E 2 , immunoreactive α -inhibin, and P, as well as the secretion of immunoreactive α -inhibin and P by the cultured granulosa-luteal cells. Results Serum immunoreactive α -inhibin levels the day of ovum pick-up were significantly lower in group 2 compared with group 1. Incubation of cells for 96hours showed a significantly higher ability to accumulate immunoreactive α -inhibin in group 1 than 2. Human chorionic gonadotropin stimulated immunoreactive α -inhibin production after 96hours. Cells from younger women displayed a significantly higher ability to secrete P than cells from older women. Human chorionic gonadotropin was able to significantly stimulate P production in group 1. Conclusions These results confirm previous observations showing a reduced production of immunoreactive α -inhibin and steroids of ovaries from older women and suggest that a reduced cellular function, rather than a decrease in the follicular population, is the main mechanism by which these changes are produced.

Viral screening of spent culture media and liquid nitrogen samples of oocytes and embryos from hepatitis B, hepatitis C, and human immunodeficiency virus chronically infected women undergoing in vitro fertilization cycles.

OBJECTIVE To assess the presence of viral RNA or DNA sequences in spent culture media used after ovum pickup (OPU) or embryo culture and in liquid nitrogen (LN) used for oocyte or embryo vitrification in patients seropositive for human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) undergoing IVF cycles. DESIGN Descriptive study. SETTING Private university-affiliated IVF center. PATIENT(S) Twenty-four women who underwent controlled ovarian stimulation for oocyte vitrification or IVF/ET. A total of 6, 11, and 6 patients were seropositive for HIV, HCV, and HBV, respectively, whereas 1 patient showed a coinfection with HCV and HBV. Seven patients presented positive blood viral load (HIV, n = 1; HBV, n = 1; HCV, n = 5). Sixty-three samples were analyzed: follicular fluid, n = 3; spent culture media, n = 33 (20 after OPU and 13 after embryo culture); and LN, n = 27 (14 and 10 after oocyte and embryo vitrification; and 3 LN storage tank samples). INTERVENTION(S) Ovum pickup, oocyte and/or embryo culture, and/or vitrification by the Cryotop open device. Reverse transcription-polymerase chain reaction analysis was performed for viral screening. MAIN OUTCOME MEASURE(S) Detection of viral sequences of HIV, HCV, and HVB. RESULT(S) All the samples analyzed tested negative for the detection of viral RNA or DNA sequences. CONCLUSION(S) We have not detected viral sequences after culture and vitrification of oocytes/embryos from HIV-, HBV-, and HCV-seropositive patients. These findings represent good evidence of the lack of risk of cross-contamination among seropositive patients, even using an open device for vitrification.

Glucocorticoid treatment decreases sera embryotoxicity in endometriosis patients

OBJECTIVE To investigate the effect of short-term glucocorticoid administration on embryotoxicity of sera from infertile patients with mild to moderate endometriosis. DESIGN Prospective longitudinal study. SETTING, PATIENTS Eight infertile patients with mild to moderate endometriosis and a control group of eight infertile patients with tubal infertility were selected on the basis of laparoscopic examination. INTERVENTIONS Basal (B) serum collection and day 1 (T1), day 3 (T2), day 6 (T3), and day 12 (T4) serum drawn after a 3-day glucocorticoid treatment in endometriosis patients. MAIN OUTCOME MEASURE Embryotoxicity of endometriosis sera, before and after glucocorticoid treatment, was investigated using a bioassay performed on two-cell mouse embryos. Interleukin 1 alpha (IL-1 alpha) and antismooth muscle, antimitochondrial, and antinuclear autoantibodies were also tested in these sera. RESULTS At 50% concentration, endometriosis serum is embryotoxic in comparison with control; 0% versus 61% of the embryos reached the blastocyst stage at 72 hours, respectively (basal versus control, P less than 0.001). However, this embryotoxicity significantly decreases 12 days after glucocorticoid treatment in comparison with untreated sera; 32.4% versus 0% of the embryos reached blastocyst stage at 72 hours, respectively (T4 versus basal, P less than 0.001), although they did not reach nontoxic levels (greater than 50%). Interleukin 1 alpha was undetectable in all samples analyzed. In endometriosis sera, antismooth muscle antibody was detected. CONCLUSIONS At 50% concentration, serum from infertile patients with minimal to moderate endometriosis appears to be embryotoxic to the in vitro development of two-cell mouse embryos. However, this embryotoxicity significantly decreases 12 days after a 3-day treatment with glucocorticoids.

Presence of Fas-Fas Ligand System and Bcl-2 Gene Products in Cells and Fluids from Gonadotropin-Stimulated Human Ovaries1

Abstract Apoptosis, or programmed cell death, is an important mechanism for the regulation of embryonic development and tissue homeostasis. It is coordinated by a number of molecules including the Fas-Fas ligand (FasL) system and bcl-2. The purpose of this study was to characterize the expression of these molecules in human oocytes and cumulus cells from gonadotropin-stimulated human ovaries and to determine whether the presence of soluble Fas (sFas), soluble FasL, or interferon-γ in follicular fluid (FF) correlated with apoptosis in cumulus cells, oocyte maturation, and embryo quality. Levels of sFas were significantly higher in FF containing immature oocytes compared with those containing atretic oocytes (P < 0.05; FF containing mature oocytes had highly variable levels of sFas. Levels of sFas in FF did not correlate with either fertilization, embryo quality resulting from fertilized oocytes, or apoptosis rate in cumulus cells. Fas was expressed in both unfertilized oocytes and cumulus cells, whereas FasL expression was not usually detected in these cell types. Messenger RNA for bcl-2 was detectable in both freshly isolated oocytes and cumulus cells but was not demonstrable following 24 h of culture that coincided with a significant increase of apoptosis in cumulus cells. Our results indicate that soluble forms of the Fas-FasL system are present in FF from gonadotropin-stimulated human ovaries and suggest that this system may play a role in preventing oocyte atresia during folliculogenesis but is probably not important for apoptotic events in cumulus cells and oocytes after fertilizaton failure. Apoptosis in this case may be facilitated by the downregulation of bcl-2. Further studies on the expression of these molecules in follicles containing atretic oocytes and immature oocytes are needed to confirm this new hypothesis.

Hormonal and molecular characterization of follicular fluid, cumulus cells and oocytes from pre-ovulatory follicles in stimulated and unstimulated cycles

BACKGROUND The use of ovarian stimulation, to stimulate a multi-follicular response for assisted reproduction treatments, may force the production of oocytes from follicles that do not reach optimal maturation, possibly yielding oocytes that are not fully competent. The present study aimed to define the follicular environment and oocyte competence of unstimulated pre-ovulatory follicles, to compare it with that of similar-sized stimulated follicles. For this purpose, we analyzed the follicular hormonal milieu, the oocyte meiotic spindle, the embryo development and the cumulus cells gene expression (GE) profiles. METHODS AND RESULTS The study population was divided in two groups: (i) 42 oocyte donors undergoing unstimulated cycles and (ii) 18 oocyte donors undergoing controlled ovarian stimulation cycles (COS). Follicular fluid was analyzed to quantify the concentrations of estradiol (E2), progesterone (P), FSH, LH, testosterone (T) and androstendione (Δ4). T was higher in the COS group, while Δ4, E2 and LH were significantly higher in unstimulated cycles. The cumulus oophorus cells (CC) surrounding the oocyte were removed and their GE profiles were analyzed with microarrays. There were 18 differentially expressed genes in CC: 7 were up-regulated and 11 were down-regulated in the COS cycles. The microarray was validated by qRT-PCR. The analysis of spindle structure revealed no significant differences between the groups, except for the parameter of length which presented differences. The fertilization ability and embryo morphology on Days 2, 3 and 4 did not show any significant differences between groups. CONCLUSIONS The use of ovarian stimulation induces changes in the follicular fluid and in CC GE that may affect immune processes, meiosis and ovulation pathways. Although these differences do not seem to relate to early-stage embryo morphology, the implications of some of the molecules, especially ALDH1A2, CTSL and ZNF33B at the CC level, deserve to be addressed in future studies.

Revised guidelines for good practice in IVF laboratories (2015)

STUDY QUESTION Which recommendations can be provided by the European Society of Human Reproduction and Embryology Special Interest Group (ESHRE SIG) Embryology to support laboratory specialists in the organization and management of IVF laboratories and the optimization of IVF patient care? SUMMARY ANSWER Structured in 13 sections, the guideline development group formulated recommendations for good practice in the organization and management of IVF laboratories, and for good practice of the specific procedures performed within the IVF laboratory. WHAT IS KNOWN ALREADY NA. STUDY DESIGN, SIZE, DURATION The guideline was produced by a group of 10 embryologists representing different European countries, settings and levels of expertise. The group evaluated the document of 2008, and based on this assessment, each group member rewrote one or more sections. Two 2-day meetings were organized during which each of the recommendations was discussed and rewritten until consensus within the guideline group was reached. After finalizing the draft, the members of the ESHRE SIG embryology were invited to review the guideline. PARTICIPANTS/MATERIALS, SETTING, METHODS NA. MAIN RESULTS AND THE ROLE OF CHANCE The guideline provides recommendations on the general organization of an IVF laboratory (staffing and direction, quality management, laboratory safety), and on the specific aspects of the procedures performed in IVF laboratories (Identification of patients and traceability of their reproductive cells, consumables, handling of biological material, oocyte retrieval, sperm preparation, insemination of oocytes, scoring for fertilization, embryo culture and transfer, and cryopreservation). A last section provides recommendations regarding an Emergency plan for IVF laboratories. LIMITATIONS, REASONS FOR CAUTION Evidence on most of the issues described is scarce, and therefore it was decided not to perform a formal search for and assessment of scientific evidence. However, recommendations published in the EUTCD and relevant and recent documents, manuals and consensus papers were taken into account when formulating the recommendations. WIDER IMPLICATIONS OF THE FINDINGS Despite the limitations, the guideline group is confident that this document will be helpful to directors and managers involved in the management and organization of IVF laboratories, but also to embryologists and laboratory technicians performing daily tasks. STUDY FUNDING/COMPETING INTERESTS The guideline was developed and funded by ESHRE, covering expenses associated with the guideline meetings. The guideline group members did not receive payment. Dr Coticchio reports speakers fees from IBSA and Cook, outside the submitted work; Dr Lundin reports grants from Vitrolife, personal fees from Merck Serono, non-financial support from Unisense, outside the submitted work; Dr. Rienzi reports personal fees from Merck Serono, personal fees from MSD, grants from GFI, outside the submitted work; the other authors had nothing to disclose. TRIAL REGISTRATION NUMBER NA.

Reduced oxygen tension improves embryo quality but not clinical pregnancy rates: a randomized clinical study into ovum donation cycles.

OBJECTIVE To investigate the effect of low O2 tension during in vitro culture in terms of ongoing pregnancy rates in ovum donation cycles. DESIGN Randomized trial. SETTING Private university-affiliated IVF center, university-based hospital. PATIENT(S) A total of 1,125 cycles of ovum donation. INTERVENTION(S) Embryo culture in an atmosphere of 5.5% CO2, 6% O2, and 88.5% N2 versus a dual-gas system of 5.5% CO2 in air. MAIN OUTCOME MEASURE(S) Ongoing clinical pregnancy rates per intention-to-treat (ITT) patients. RESULT(S) The use of low O2 tension achieved a 41.3% ongoing pregnancy rate per ITT compared with a 40.8% rate obtained for 5% CO2 in air. The mean number of blastomeres and the percentage of top-quality embryos were significantly higher after lower O2 concentration during in vitro culture (7.1 ± 3.6 and 28.6% vs. 7.3 ± 8.4 and 32.1%, respectively). CONCLUSION(S) In the ovum donation cycles undergoing day-3 embryo transfers, the use of low O2 tension did not improve ongoing pregnancy rates per cycle and per transfer. However, it benefited embryo quality, demonstrating the potential negative impact of high O2 tension on the in vitro embryo development.

A multicenter prospective study to assess the effect of early cleavage on embryo quality, implantation, and live-birth rate

OBJECTIVE To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates. DESIGN Prospective cross-sectional study. SETTING Multicenter study. PATIENT(S) Seven hundred embryo transfers and 1,028 early-stage human embryos. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Implantation according to the presence of EC and embryo quality. RESULT(S) The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage. CONCLUSION(S) At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation.

The dynamics of in vitro maturation of germinal vesicle oocytes

OBJECTIVE To evaluate the dynamics of the nuclear maturation (NM) of in vitro-matured (IVM) oocytes and to determine the most favorable duration of meiosis II (MII) arrest in relation to the normal activation response. DESIGN Experimental. SETTING University-affiliated infertility clinic. PATIENT(S) Donated immature germinal vesicle oocytes (GV). INTERVENTION(S) The GV underwent spontaneous IVM and the dynamics of NM studied by real-time monitoring. The IVM oocytes were parthenogenetically activated at different MII arrest points and their response assessed. MAIN OUTCOME MEASURE(S) Moment of GV breakdown; extrusion of the first polar body; duration of MI and MII arrest; activation rate (AR) and type. RESULT(S) Two GV populations-early (E-IVM, 18.4 ± 2.7 hours) and late (L-IVM, 26.3 ± 3.8 hours) maturing-were defined according to the time required for extrusion of the first polar body. Significantly more E-IVM than L-IVM exhibited a normal activation response (61.3% vs. 34.6%), but AR were similar (average, 88.6%) in both groups. Duration of the GV stage differed between the two groups, but MI arrest (14.0 ± 0.3 hours) was constant. The E-IVM arrested at MII for at least 4.3 hours displayed significantly lower AR and similar normal activation rates (61.3%) to E-IVM arrested for a shorter time (83.9% vs. 100%). The L-IVM displayed a similar AR (80.8%), but lower normal activation rates than E-IVM (34.6%), regardless of when activation took place. CONCLUSION(S) The success of IVM depends on the NM timing rather than on the length of MII arrest.

Effect of a sharp serum oestradiol fall before HCG administration during ovarian stimulation in donors

The current study evaluated how a sudden fall in serum oestradiol during ovarian stimulation in donors affects recipient outcome. After the assessment of pregnancy rate in cases of oestradiol falls of <10 or > or =10% (57.0 versus 45.6%), <20 or > or = 20% (55.2 versus 44.9%), <25 or > or =25% (57.2 versus 41.2%), and < 30 or > or =30% (57.1 versus 32.0%; P < 0.05), a significantly lower pregnancy rate was observed when the fall was > or =30%. Therefore, the study group (n = 25) included recipients who received oocytes from donors with a fall of > or =30%, and the control group included patients (n = 197) in which the fall in oestradiol was <30% and all cases with no fall in oestradiol concentrations. Pregnancy rates in both groups were 32.0 versus 57.1%; P < 0.05. The number of morphologically normal oocytes was similar (14.2 versus 18.1%) and good quality embryos was lower (8.0 versus 21.0%; P < 0.05) for study group. This seems related to a lower capability of the embryos to implant (15.2 versus 37.4%; P < 0.001). These data indicate that a fall of > or =30% in serum oestradiol concentration during ovarian stimulation in donors negatively affects pregnancy rates and embryo quality in recipients. In these cases, cycle cancellation should be considered.