Serum miR-181d-5p levels in response to controlled ovarian stimulation: predictive value and biological function.

The response to controlled ovarian stimulation (COS) for in vitro fertilization (IVF) varies dramatically from one patient to another, affecting success rates. A previous large-scale study identified increased serum miR-181d-5p levels in patients with high response to COS prior to stimulation. We aim to evaluate whether the expression of miR-181d-5p differs according to the ovarian response to COS in women undergoing intracytoplasmic sperm injection (ICSI) cycles. Samples collected prior to COS for ICSI were split into three groups depending on the ovarian response to COS: poor response (PR), <4 oocytes retrieved (n=25); normal response (NR), ≥8 and ≤12 oocytes retrieved (n=21); and high response (HR), >25 oocytes retrieved (n=20). miR-181d-5p serum levels were compared among experimental groups. miR-181d-5p levels were increased in the HR group when compared to the PR (p=0.0001) and NR groups (p=0.0079). miR-181d-5p levels correlated with the number of aspirated follicles (p<0.0001), retrieved oocytes (p<0.0001), and mature oocytes (p=0.0002). Increased miR-181d-5p levels independently predict a high response (p=0.006), with Positive and Negative Predictive Values of 66.7% and 69.4%, respectively. miR-181d-5p was also detected in the ovarian tissue in a mouse model. Moreover, computational analysis of miR-181d-5p predicted targets and promoter region suggested that this miRNA might be involved in the regulation of key signaling pathways and biological processes for female reproductive biology. In conclusion, miR-181d-5p is a promising circulating predictor of high stimulation and potential mediator of the hypothalamus-pituitary-gonad axis, providing opportunities for the individualization of COS protocols.

Is there an association between artificial sweetener consumption and assisted reproduction outcomes?

Previous studies have suggested an association between high intake of sweetened beverages and a number of adverse health outcomes. In this cross-sectional study, we investigated the association between daily consumption of sweetened soft drinks or coffee and the outcome of intracytoplasmic sperm injection (ICSI) treatment. Patients (n = 524) were interviewed by a nutritionist before ICSI treatment, using a food frequency questionnaire. Regression analysis showed that consumption of ≥3 servings of regular soft drinks or any amount of diet soft drinks was associated with oocyte dysmorphism, diminished embryo quality on days 2 and 3 of culture, and a mild effect on blastocyst formation, implantation and pregnancy rate. Consumption of artificially sweetened coffee was negatively associated with embryo quality on days 2 and 3. However, consumption of coffee or soft drinks was not associated with the odds of live birth. Even so, patients should be advised about the potential negative effects of sugar and artificial sweeteners before attempting infertility treatment. This study is limited by the use of a non-validated food frequency questionnaire, lack of information on quantity of sweeteners consumed, and lack of data on glucose levels in blood serum or follicular fluid. Further investigation is warranted.

FSH dose to stimulate different patient' ages: when less is more.

OBJECTIVE: To determine the effect of FSH doses on intracytoplasmic sperm injection (ICSI) outcomes according to the age of the patient. METHODS: Patients undergoing controlled ovarian stimulation (COS) for ICSI cycles in a university-affiliated in vitro fertilization center were split into age groups: ≤35 y.o. (n=1523); >35 and ≤38 y.o. (n=652); >38 and ≤40 y.o. (n=332); and >40 y.o. (n=370). The effect of FSH dose on COS, laboratorial and clinical outomes was determined by linear regression models. RESULTS: The FSH dose didn't affect the ovarian response in terms of total number of follicles, retrieved oocytes and mature oocytes within the age groups, but we found that the lower the age, the lower the FSH dose needed per oocyte retrieved. In the group of patients ≤35 y.o., we also found a positive effect of the FSH dose on oocyte yield. Despite that, for patients ≤38 y.o. there was a negative effect of the FSH dose on embryo quality and blastocyst formation rate, and an increase in the cycle's cancelation rate. In patients ≥39 y.o., there were no effects of the FSH doses on the analysed variables. CONCLUSIONS: Ovarian stimulation with high doses of FSH is not recommended in younger women (≤38 y.o.), once we found a decrease in embryo quality and an increase in cycle's cancelation rate. Mild ovarian stimulation protocols may be more appropriate; however, it may not be applicable for women in advanced age, since a higher FSH dose is needed for oocyte retrieval in these patients.

miR-142-3p as a biomarker of blastocyst implantation failure - A pilot study.

OBJECTIVE: This study aims to find whether microRNAs (miRNAs) detected in the culture medium of embryos produced in vitro could be potential biomarkers of embryo implantation. METHODS: Culture media samples from 36 embryos, derived from patients undergoing intracytoplasmic sperm injection (ICSI) in a private university-affiliated IVF center, were collected between January/2015 and November/2015. Samples were collected on day three and embryo transfers were performed on day five and all embryos reached the blastocyst stage. Samples were split into groups according to the embryo implantation result: Positive-Implantation-Group (n=18) or Negative-Implantation-Group (n=18). For the first analysis, samples were pooled in three sets for each group (6-7 spent media per pool). MicroRNAs were extracted from spent media and cDNA was synthesized. C. elegans miR-39 was used as RNA spike-in to normalize the gene expression analysis. The expression of microRNAs into the spent media from the Positive-Implantation-Group was compared with those from the Negative-Implantation-Group. A set of seven miRNAs (miR-21, miR-142-3p, miR-19b, miR-92a, miR-20b, miR-125a and miR148a) selected according with the literature, was tested. To check whether miRNAs could be detected in individual samples of culture media, in a second analysis, ten more samples were tested for miR-21 and miR-142-3p. RESULTS: From the sevens tested miRNAs, a significant increased expression of miR-142-3p could be noted in the Negative-Implantation-Group (P<0.001). For other three miRNAs (miR-21, miR-19b and miR-92a) a difference in expression was observed, however it did not reach a statistical significance. In addition, when ten non-redundant samples were tested to check if miRNAs could be detected in individual samples of culture media, the highly specific amplification of mature miRNAs, including miR-142-3p, could be noted. CONCLUSION: Our findings suggest that miR-142-3p, previously described as a tumor suppressor and cell cycle inhibitor, may be a potential biomarker of blastocyst implantation failure. The identification of miRNAs on individual culture medium samples offers unique opportunities for non-invasive early diagnosis of blastocyst implantation.

The impact of the embryo quality on the risk of multiple pregnancies.

The aim of the present study was to determine the chance of pregnancy and the risk of multiple pregnancies taking into account the number and quality of transferred embryos in patients >36 years old or ≤36 years old. For this study, 1497 patients undergoing intra-cytoplasmic sperm injection (ICSI) cycles in a private assisted reproduction centre were split into groups according to the number and quality of the transferred embryos on the third or fifth day of development. The pregnancy rate and multiple pregnancy rate were compared between the embryo quality groups in patients <36 years old or ≥36 years old. In patients <36 years old, for the day 3 embryo transfer, no significant difference was noted in the pregnancy rate when the groups were compared. However the multiple pregnancy rate was increased by the transfer of an extra low-quality embryo (17.1 versus 28.2%, P = 0.020). For day 5 embryo transfer, the transfer of an extra blastocyst significantly increased the pregnancy rate (36.0 versus 42.4%, P < 0.001) and the multiple pregnancy rate (4.4 versus 16.9%, P < 0.001). In older patients, no significant difference was noted in the pregnancy rate when the groups were compared. However, when an extra low-quality embryo was transferred, a significantly increased rate of multiple pregnancies was observed for day 3 (18.2 versus 26.4%, P = 0.049) and day 5 embryo transfers (5.2 versus 16.1%, P < 0.001). In conclusion, the transfer of an extra low-quality embryo may increase the risk of a multiple pregnancy. In younger patients, the transfer of an extra low-quality blastocyst may also increase the chance of pregnancy.

The combination of pronuclear and blastocyst morphology: a strong prognostic tool for implantation potential.

PURPOSE: To (i) investigate a possible association between different features of pronuclear (PN) morphology and different features of blastocyst morphology, (ii) evaluate the combination of PN and blastocyst morphologies as a predictive factor for ICSI outcomes and (iii) identify possible contributing factors to poor PN morphology. METHODS: This study included 908 normally fertilised zygotes reaching full blastocyst stage, obtained from 350 patients undergoing ICSI cycles, in which the implantations rates were 0 % or 100 %. The influence of PN morphology on blastocyst morphology and on the rates of pregnancy and miscarriage was investigated. Embryos were graded and split into three groups, taking into consideration both the PN and the blastocyst status. The pregnancy rate was compared among these groups. RESULTS: Inner cell mass (ICM) alterations were correlated with the number of nucleolar precursor bodies (NPB), while trophectoderm alterations were correlated with the size of the pronuclei and the distribution of the NPB. The distribution of the NPB had an impact on the chances of pregnancy. A significant difference was observed among the groups regarding the pregnancy rate. The maternal age, number of aspirated follicles and number of retrieved oocytes influenced the incidence of PN defects. CONCLUSIONS: These findings suggest that a lower oocyte yield may lead to higher-quality PN zygotes. In addition, different PN features may influence further embryo development, especially the quality of the blastocyst. Moreover, the association between PN and blastocyst morphology may be used as a prognostic tool for implantation.

Influence of oocyte dysmorphisms on blastocyst formation and quality.

OBJECTIVE: To identify the association between oocyte dysmorphisms and blastocyst developmental competence. DESIGN: Case-control study. SETTING: Private assisted reproduction center. PATIENT(S): This study included 5,516 embryos, which were obtained from 605 patients undergoing intracytoplasmic sperm injection cycles in which ET was performed on day 5 of embryo development. INTERVENTION(S): Intracytoplasmic sperm injection and ET. MAIN OUTCOME MEASURE(S): The morphologic characteristics of the blastocysts, namely the degree of blastocoel expansion and hatching status, the size and compactness of the inner cell mass (ICM), and the cohesiveness and number of trophectoderm cells, were correlated with oocyte dysmorphisms. RESULT(S): Of the 5,516 embryos evaluated, 2,920 (52.9%) reached the blastocyst stage. The blastocysts' degree of expansion and hatching status were decreased by the presence of aggregates of smooth endoplasmic reticulum clusters, large perivitelline space, and shape abnormalities. The presence of a large perivitelline space also decreased the ICM and trophectoderm cell quality. Aggregates of smooth endoplasmic reticulum clusters also decreased the ICM quality, pregnancy rate, and the miscarriage rate. CONCLUSION(S): The results presented here suggest that the individual identification of oocyte dymorphisms may be a prognostic tool for blastocyst development and quality. However, the results do not diminish the importance of cleavage-stage embryo morphology assessment to predict blastocyst development competence.

Relevance of assisted hatching in an oocyte donation programme using egg cryobanking: a prospective randomised study.

OBJECTIVE: The objective of this prospective randomised study was to evaluate if there is an improvement in clinical outcomes when assisted hatching (AH) is performed in embryos derived from vitrified/warmed oocytes in an ovum donation programme using egg cryobanking. STUDY DESIGN: Sixty oocyte recipients in a donation programme using egg cryobanking were randomly allocated to the assisted hatched (AH, n=30) or control group (n=30). The pregnancy and implantation rates were compared between the groups. RESULTS: A total of 288 vitrified oocytes were warmed for the 60 recipients. Of the 288 vitrified oocytes, 94.8% survived. All surviving oocytes were sperm injected, and 83.5% underwent fertilisation. There were 172 good-quality embryos selected for transfer. The total pregnancy rate was 40%. The pregnancy rate did not differ between the AH and control groups (43.3% and 33.3%, respectively, p=0.1967), but AH resulted in a higher implantation rate (31.6% vs. 18.4%, p=0.0206). CONCLUSION: Our study demonstrates the effectiveness of AH in embryos derived from warmed oocytes. Our results also suggest that oocyte cryopreservation can be considered as a tool for providing highly successful outcomes in an egg donor programme.

Contributing factors for the incidence of aneuploidy in older patients undergoing intracytoplasmic sperm injection cycles.

PURPOSE: To evaluate different factors that might affect the incidence of embryo aneuploidy in intracytoplasmic sperm injection cycles (ICSI). METHODS: One hundred and ninety ICSI cycles in conjunction with preimplantation genetic screening (PGS) were included. The influence of the following variables on the aneuploidy incidence was evaluated: (i) maternal and (ii) paternal ages, (iii) dose of FSH administered, (iv) dose of FSH per number of retrieved matured oocytes (FSH/MII), (v) serum 17ß-oestradiol levels on the ovulation trigger day, (vi) aspirated follicles and (vii) retrieved oocytes. RESULTS: A total of 440 embryos were successfully biopsied, of which 240 were considered euploid and 200 were considered aneuploid. The paternal age (Slope: 0.2, p = 0.372), total dose of FSH (Slope: 0.2, p = 0.218), FSH/MII (Slope: 0.1, p = 0.296) and 17ß-oestradiol levels (Slope: 0.2, p = 0.378) were not correlated with the presence of aneuploidy. However, the maternal age (Slope: 1.7, p < 0.01), aspirated follicles (Slope: 1.9, p < 0.01) and retrieved oocytes (Slope: 2.6, p < 0.01) were negatively correlated with the incidence of aneuploidy. CONCLUSIONS: Even in older patients, lower oocyte yields may represent a more appropriate response to ovarian stimulation, allowing the most competent follicles and oocytes to develop and thereby reducing the occurrence of embryo aneuploidy.

A chromosome 19 locus positively influences the number of retrieved oocytes during stimulated cycles in Brazilian women.

PURPOSE: To evaluate if several genetic loci that are associated with variation in normal menopause age and early menopause can account for a poor response to controlled ovarian stimulation. METHODS: A total of 71 patients age ≤35 years old who were undergoing intracytoplasmic sperm injection were genotyped for four genetic variants that are associated with normal variation in menopausal age and early menopause. The patients were divided into two groups based upon treatment response: a poor responder group (PR group, n = 21) and a normal responder group (NR group, n = 50). The genetic variants rs244715, rs9379896, rs4806660 and rs16991615 were analyzed. RESULTS: There was no significant difference in the incidence of the genetic variants between the NR and PR group. The risk allele for the chromosome 19 variant (rs4806660) demonstrated a protective effect for a poor ovarian response. The presence of a risk allele was associated with an increased response to COS, which resulted in an elevated number of follicles (Coef: 2.54, P = 0.041) and retrieved oocytes (Coef: 1.41, P = 0.041). CONCLUSIONS: The genetic variants rs244715, rs9379896, rs4806660 and rs16991615 are not risk factors for poor ovarian response in Brazilian women. In contrast, rs4806660 is associated with higher number of follicles and retrieved oocytes. rs4806660 may be associated with an increased response to gonadotrophin stimulation in this population.

Food intake and social habits in male patients and its relationship to intracytoplasmic sperm injection outcomes.

OBJECTIVE: To investigate the influence of the male partner's lifestyle, including eating and social habits, on semen quality and intracytoplasmic sperm injection (ICSI) success. DESIGN: Observational study. SETTING: Private fertility clinic. PATIENT(S): Two hundred fifty male patients undergoing ICSI cycles. INTERVENTION(S): We recorded dietary and social habits using a food frequency questionnaire adapted to meet specific study objectives. Evaluation of semen parameters and ICSI outcomes were performed. MAIN OUTCOME MEASURE(S): Frequency of intake of food items and social habits were registered on a scale with five categories ranging from no consumption to repeated daily consumption. RESULT(S): The sperm concentration was negatively influenced by body mass index (BMI) and alcohol consumption and was positively influenced by cereal consumption and the number of meals per day. The sperm motility was also negatively influenced by BMI, alcohol consumption, and smoking habit, whereas it was positively influenced by the consumption of fruits and cereals. The consumption of alcohol had a negative influence on the fertilization rate. The consumption of red meat as well as being on a weight loss diet had a negative impact on the implantation rate. In addition, the consumption of red meat and being on a weight loss diet had an effect on the pregnancy chance. CONCLUSION(S): Couples seeking assisted reproduction treatments must be advised about the drastic effect of both the male and female lifestyle on treatment success.

Intracytoplasmic morphologically selected sperm injection benefits for patients with oligoasthenozoospermia according to the 2010 World Health Organization reference values.

The comparison between the outcomes of intracytoplasmic morphologically selected sperm injection performed in couples with male factor infertility according to the World Health Organization guidelines from 1999 and 2010 was the objective of this study. Our results suggest that the sperm selection under high magnification results in improved treatment outcomes in patients with oligoasthenoteratozoospermia, according to the new World Health Organization guidelines.

Morphological nuclear integrity of sperm cells is associated with preimplantation genetic aneuploidy screening cycle outcomes.

OBJECTIVE: To examine the effect of sperm morphology on embryo development at the chromosomal level. DESIGN: Prospective study. SETTING: Assisted fertilization center. PATIENT(S): Couples who underwent IVF-PGS cycle, as a result of advanced maternal age, were randomly allocated into two groups: intracytoplasmic sperm injection (ICSI; n = 60) or intracytoplasmic morphologically selected sperm injection (IMSI; n = 60). INTERVENTION(S): IVF in conjunction with preimplantation genetic screening (PGS). MAIN OUTCOME MEASURE(S): Sperm nuclear morphology at high-magnification ICSI and incidence of aneuploidy in derived embryo. RESULT(S): There was a significantly increased incidence for sex chromosome aneuploidy in ICSI embryos when compared with IMSI embryos (23.5% vs. 15.0%, respectively). High-magnification sperm selection was associated with a significantly lower risk of sex chromosome abnormalities (odds ratio [OR], 0.57; confidence interval [CI], 0.37-0.90). The incidence of chaotic embryos was also significantly higher with the ICSI procedure (27.5% vs. 18.8%), while the IMSI procedure was associated with a significantly lower risk of chaotic embryos (OR, 0.64; CI, 0.43-0.96). Moreover, the cycle cancellation rate was significantly higher in ICSI cycles (11.8% vs. 2.5%). High-magnification sperm selection was a significant predictor of the likelihood of cycle cancellation (OR, 0.26; CI, 0.11-0.62). CONCLUSION(S): Spermatozoa free of nuclear morphological malformations were found to be significantly associated with the lower incidence of aneuploidy in derived embryos, resulting in lower rates of cycle cancellation.