E. Labarta

Endometrial receptivity is affected in women with high circulating progesterone levels at the end of the follicular phase: a functional genomics analysis

BACKGROUND Elevated serum progesterone levels at the end of the follicular phase in controlled ovarian stimulation (COS) leads to a poorer ongoing pregnancy rate in IVF cycles due to reduced endometrial receptivity. The objective of this study was to use microarray technology to compare endometrial gene expression profiles at the window of implantation according to the levels of circulating progesterone. METHODS For this prospective cohort study, microarray data were obtained from endometrial biopsies from 12 young healthy oocyte donors undergoing COS with pituitary suppression by either gonadotrophin-releasing hormone (GnRH) agonists or antagonists, and recombinant FSH. On the day of recombinant chorionic gonadotrophin (rCG) administration, six women had serum progesterone levels (P) >1.5 ng/ml (study group) and six had serum P levels <1.5 ng/ml (control group). Endometrial samples were collected using a Pipelle catheter 7 days after the rCG injection. RESULTS Using the parametric test, we identified 140 genes significantly dysregulated (64 up- and 76 down-regulated) in the study group endometria compared with the control endometria, regardless of the GnRH analogue employed. These genes are related to cell adhesion, developmental processes, the immune system and others, which are all required for normal endometrial function development. Of the 25 gene targets previously proposed as markers for endometrial receptivity, 13 appeared over-regulated in the study group. CONCLUSIONS Our results reveal that elevated progesterone levels on the day of rCG administration can induce significant alterations in the gene expression profile of the endometrium.

Impact of luteinizing hormone administration on gonadotropin-releasing hormone antagonist cycles: an age-adjusted analysis

OBJECTIVE To analyze the impact of LH administration on cycle outcome in ovarian stimulation with GnRH antagonists. DESIGN Randomized, open-label, controlled trial performed in two age subgroups. Recombinant (r) FSH versus rFSH + rLH administration was compared. SETTING University-affiliated private infertility clinic. PATIENT(S) Up to 35 years old (n = 380) and aged 36 to 39 years (n = 340), undergoing their first or second IVF cycle. INTERVENTION(S) Recombinant LH administration since stimulation day 1. MAIN OUTCOME MEASURE(S) Implantation rate, ongoing pregnancy rate. RESULT(S) In the young population, implantation rates were similar: 27.8% versus 28.6%, odds ratio (OR) 1.03 (95% confidence interval [CI] 0.73-1.47), as was the ongoing pregnancy rate per started cycle: 37.4% versus 37.4%, OR 1.0 (95% CI 0.66-1.52). In older patients, the implantation rate was significantly higher in the rFSH + rLH group: 26.7% versus 18.6%, OR 1.56 (95% CI 1.04-2.33). Ongoing pregnancy rates per started cycle were 33.5% versus 25.3%, OR 1.49 (95% CI 0.93-2.38). CONCLUSION(S) Recombinant LH administration significantly increased the implantation rate in patients aged 36 to 39 years. A clinically relevant better ongoing pregnancy rate per started cycle was observed, although the difference was not statistically significant. Patients younger than 36 years do not obtain any benefit from rLH administration.

Highly purified hMG versus recombinant FSH in ovarian hyperstimulation with GnRH antagonists—a randomized study

BACKGROUND Highly purified hMG (hp-hMG) has recently shown better cycle outcome than the recombinant FSH (rFSH) when compared in GnRH agonist long protocol cycles. However, they have not yet been compared in GnRH antagonist cycles. METHODS A RCT comparing the ongoing pregnancy rate (primary end-point) in 280 patients undergoing IVF/ICSI after stimulation with hp-hMG or rFSH controlled with a GnRH antagonist. RESULTS No significant differences were observed between hp-hMG and rFSH in terms of the ongoing pregnancy rate per started cycle (35.0 versus 32.1%, respectively; P = 0.61); relative risk: 1.09 (95% confidence interval: 0.78-1.51; risk difference: 2.9%). No differences were observed for implantation, clinical pregnancy and pregnancy loss rates. More oocytes were obtained from patients receiving rFSH then hMG (14.4 +/- 8.1 versus 11.3 +/- 6.0, respectively; P = 0.001). Estradiol was higher at the end of stimulation in the hp-hMG group (P = 0.02), whereas progesterone was higher in patients stimulated with rFSH (P < 0.001). CONCLUSIONS A similar outcome was observed for hp-hMG and rFSH when used for stimulation in GnRH antagonist cycles. However, some differences were found in ovarian response in terms of oocyte yield and hormonal profile. Clinical Trials.gov TRIAL REGISTRATION NUMBER NCT00669786.

Endometrial gene expression in the window of implantation is altered in obese women especially in association with polycystic ovary syndrome.

OBJECTIVE To determine whether luteal phase endometrial transcriptome is altered in obese women during the window of implantation (WOI), considering the presence of infertility, fat distribution and association with polycystic ovary syndrome (PCOS). DESIGN Prospective study. SETTING University-affiliated infertility clinic, between May 2007 and March 2009. PATIENT(S) One control group of women with normal weight (n=4), and four study groups of obese women (n=6 each one) according to the association with infertility, PCOS, and ovarian stimulation. INTERVENTION(S) The endometrium was biopsied 7 days after LH surge or hCG administration in 28 women. MAIN OUTCOME MEASURE(S) Endometrial gene expression during the WOI. RESULT(S) One hundred and fifty-one genes were dysregulated in obese groups compared with controls. This dysregulation was more pronounced when infertility was associated. The biologic processes of these genes belonged mainly to development and regulation of different biological functions such as transcription and biosynthesis. The molecular functions overrepresented were transcription and peptide receptor activity. The endometrium of obese women with PCOS showed dysregulated genes related to biologic processes such as development, morphogenesis, and the immune system, as well as different molecular functions such as protein binding, binding, growth factor activity, and carboxylic acid transmembrane transporter activity. Some of these genes have been previously related to implantation and unexplained infertility. CONCLUSION(S) Obese women present a different endometrial gene expression than controls during the WOI, which is more pronounced when infertility or polycystic ovary syndrome are associated.

Prospective cohort study in high responder oocyte donors using two hormonal stimulation protocols: impact on embryo aneuploidy and development

BACKGROUND Ovarian stimulation regimens for in vitro fertilization seem to have a deleterious effect on oocyte quality and embryo aneuploidy in a dose-dependent manner. This study aims to test the influence of gonadotrophin doses on embryo aneuploidy rates. METHODS A total of 32 young oocyte donors with a high response to ovarian stimulation, were included in the study. Two subsequent stimulation treatments were performed in each donor: first, a standard dose cycle using a 225 IU starting dose of recombinant FSH (r-FSH) and secondly, a reduced dose cycle with a starting dose of 150 IU r-FSH. In both cycles, GnRH agonist co-treatment was used for down-regulation. Ovarian response, embryo development and aneuploidy for chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y were the main outcomes of the study. RESULTS A total of 22 donors completed both treatments with different gonadotrophin doses. In the remaining 10 donors, the reduced dose cycle was cancelled due to low ovarian response. In those donors who completed both regimens, significant increases in rates of fertilization and chromosomally normal blastocysts were observed in the reduced dose cycle. No differences were observed in pregnancy and implantation rates in recipients who received oocytes from standard and reduced doses cycles. CONCLUSIONS Despite the limited numbers in our study, we can conclude that in high responder donors, a decrease in the gonadotrophin dose could improve fertilization rates and embryo quality. However, due to the reduced oocyte numbers with lower doses, a similar reproductive outcome in terms of live births would be expected.

Moderate Ovarian Stimulation Does Not Increase the Incidence of Human Embryo Chromosomal Abnormalities in in Vitro Fertilization Cycles

CONTEXT A high chromosomal abnormalities rate has been observed in human embryos derived from in vitro fertilization (IVF) treatments. The real incidence in natural cycles has been poorly studied, so whether this frequency may be induced by external factors, such as use of gonadotropins for ovarian stimulation, remains unknown. DESIGN We conducted a prospective cohort study in a University-affiliated private infertility clinic with a comparison between unstimulated and stimulated ovarian cycles in the same women. Preimplantation genetic screening by fluorescence in situ hybridization was performed in all viable d 3 embryos. OBJECTIVE The primary objective was to compare the incidence of embryo chromosomal abnormalities in an unstimulated cycle and in an ulterior moderate ovarian stimulated cycle. Secondary outcome measures were embryo quality, blastocyst rate of biopsied embryos, number of normal blastocysts per donor, type of chromosomal abnormalities, and clinical outcome. RESULTS One hundred eighty-five oocyte donors were initially recruited for the unstimulated cycle, and preimplantation genetic screening could be performed in 51 of them, showing 35.3% of embryo chromosomal abnormalities. Forty-six of them later completed a stimulated cycle. The sperm donor sample was the same for both cycles. The proportion of embryos displaying abnormalities in the unstimulated cycle was 34.8% (16 of 46), whereas it was 40.6% (123 of 303) in the stimulated cycle with risk difference=5.8 [95% confidence interval (CI)=-20.6-9.0], and relative risk=1.17 (95% CI=0.77-1.77) (P=0.45). When an intrasubject comparison was made, the abnormalities rate was 34.8% (95% CI=20.5-49.1) in the unstimulated cycle and 38.2% (95% CI=30.5-45.8) in the stimulated cycle [risk difference=3.4 (95% CI=-17.9-11.2); P=0.64]. No differences were observed for embryo quality and type of chromosomal abnormalities. CONCLUSIONS Moderate ovarian stimulation in young normo-ovulatory women does not significantly increase the embryo aneuploidies rate in in vitro fertilization-derived human embryos as compared with an unstimulated cycle. Whether these results can be extrapolated to infertile patients is still unknown.

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.

Day-3 embryo metabolomics in the spent culture media is altered in obese women undergoing in vitro fertilization

OBJECTIVE To determine whether the global metabolomic profile of the spent culture media (SCM) of day-3 embryos is different in obese and normoweight women undergoing in vitro fertilization (IVF). DESIGN Prospective cohort analysis. SETTING IVF clinic. PATIENT(S) Twenty-eight young, nonsmoking women with normoweight, nonsmoking male partners with mild/normal sperm factors undergoing a first IVF attempt for idiopathic infertility, tubal factor infertility, or failed ovulation induction: obese ovulatory women (n = 12); obese women with polycystic ovary syndrome (PCOS; n = 4); normoweight ovulatory women (n = 12). INTERVENTION(S) Fifty μl of SCM collected from two day-3 embryos of each cohort. MAIN OUTCOME MEASURE(S) Metabolomic profiling via ultrahigh performance liquid chromatography coupled to mass spectrometry of SCM from a total of 56 embryos. RESULT(S) The untargeted metabolomic profile was different in obese and normoweight women. Partial least squares discriminant analysis resulted in a clear separation of samples when a total of 551 differential metabolites were considered. A prediction model was generated using the most consistent metabolites. Most of the metabolites identified were saturated fatty acids, which were detected in lower concentrations in the SCM of embryos from obese women. The metabolomic profile was similar in obese women with or without PCOS. CONCLUSION(S) The metabolomic profile in the SCM of day-3 embryos is different in normoweight and obese women. Saturated fatty acids seem to be reduced when embryos from obese patients are present. CLINICAL TRIAL REGISTRATION NUMBER NCT01448863.

The follicular hormonal profile in low-responder patients undergoing unstimulated cycles: is it hypoandrogenic?

STUDY QUESTION What is the final hormonal milieu of pre-ovulatory follicles of low-responder (LR) patients undergoing unstimulated cycles? SUMMARY ANSWER Neither androgen secretion nor LH was impaired in pre-ovulatory follicles of LR women. WHAT IS KNOWN ALREADY Therapies currently used to improve ovarian response in LR women have an impact on the final hormonal follicular milieu, and these changes are believed to be partially responsible for determining the success rate in these women. Surprisingly, as far as we know, there is no report of the final hormonal profile of LR women undergoing unstimulated cycles or evidence that follicular androgen secretion in LR women is impaired. STUDY DESIGN, SIZE AND DURATION A prospective case-control study including 94 women, 36 normal controls and 58 LR patients (19 Young ≤ 35 years LR and 39 Aged >35 years LR) from 2009 to 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS Fifty-eight LR women were divided into two groups: Young LR (age ≤ 35; n = 19) and Aged LR (ALR; age >35; n = 39). The control group (group C) comprised 36 egg donors undergoing an unstimulated cycle in our IVF unit. Serum and follicular fluid hormonal concentrations for estradiol (E₂), progesterone, testosterone and androstendione were measured. The spindle parameters of metaphase II oocytes generated from these groups were also analysed. MAIN RESULTS AND THE ROLE OF CHANCE Pre-ovulatory follicles from LR patients had similar androgenic and LH concentrations to those observed in the control group. However, higher intrafollicular concentrations of FSH and progesterone were observed in ALR. Moreover, no differences were found for the spindle evaluation of oocytes between groups by the Oosight technology. LIMITATIONS, REASONS FOR CAUTION The controls were younger and had a lower BMI than the LR women. The sample size available restricted statistical power. WIDER IMPLICATIONS OF THE FINDINGS This study suggests that the problem with LR women is not the final pre-ovulatory follicular androgen concentration since this is similar to normal responders, but in the ability to respond to controlled ovarian stimulation protocols. Therefore, efforts should be focused on long-interval androgen priming to potentially increase the recruitment of small antral follicles rather than increasing the intraovarian androgen levels within the current cycle. STUDY FUNDING/COMPETING INTEREST The present project has been supported by the R+D programme from the Generalitat Valenciana (Regional Valencian Government) IMPIVA MIDTF/2010/95. The authors have no conflict of interest to declare.

Regimen of ovarian stimulation affects oocyte and therefore embryo quality

Without any doubt the regimen used to mature multiple capable oocytes for IVF impacts IVF outcomes. Studies have indicated that the inclusion of LH activity, adjuvant agents such as growth hormone (GH), and regimens providing for simultaneous action of both LH and FSH during final oocyte maturation may have beneficial effects on IVF outcomes. Because of the difficulty in improving IVF outcomes in poor responders, the studies on GH are of particular interest. As pointed out in this review, the apparent beneficial effects of GH on oocyte competence may also apply to older women or to normal responders with reduced embryo quality. A much more difficult question is whether and how much ovarian stimulation impacts on oocyte competence. Paradoxically it seems that there are not demonstrated differences between the stimulated and the natural unstimulated cycle, whereas studies in laboratory animals and IVF patients have shown deleterious effects of higher compared with lower doses of gonadotropins. Recent studies suggest that the use of high doses of gonadotropins as an independent factor correlates negatively with the probability of live birth, whereas a high ovarian response per se is associated with better cumulative pregnancy rates, owing to the availability of more euploid and good-quality embryos. Although adjunctive use of androgens has not been discussed here, it is briefly covered in the first review of this series.