Javier Crosby

The transcriptome of human oocytes

The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis.

Identification of a novel gene set in human cumulus cells predictive of an oocyte's pregnancy potential

OBJECTIVE To identify a gene expression signature in human cumulus cells (CCs) predictive of pregnancy outcome across multiple clinics, taking into account the clinic and patient variations inherent in IVF practice. DESIGN Retrospective analysis of single human cumulus-oocyte complexes with the use of a combined microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) approach. SETTING Multiple private IVF clinics. PATIENT(S) Fifty-eight patients. Samples from 55 patients underwent qRT-PCR analysis, and samples from 27 patients resulted in live birth. INTERVENTION(S) Gene expression analysis for correlation with pregnancy outcome on individual human CCs collected immediately after oocyte retrieval. Pregnancy prediction analysis used leave-one-out cross-validation with weighted voting. MAIN OUTCOME MEASURE(S) Combinatorial expression of 12 genes in 101 samples from 58 patients. RESULT(S) We found a set of 12 genes predictive of pregnancy outcome based on their expression levels in CCs. This pregnancy prediction model had an accuracy of 78%, a sensitivity of 72%, a specificity of 84%, a positive predictive value of 81%, and a negative predictive value of 76%. Receiver operating characteristic analysis found an area under the curve of 0.763 ± 0.079, significantly greater than 0.5 (random chance prediction). CONCLUSION(S) Gene expression analysis in human CCs should be considered in identifying oocytes with a high potential to lead to pregnancy in IVF-ET.

Selection of the most common chromosome abnormalities in oocytes prior to ICSI

So far, all preimplantation genetic diagnosis (PGD) protocols in use produce results after the eggs have been fertilized. However, these approaches are not acceptable for patients with moral objections to the generation and discard of supernumerary zygotes or embryos. In these circumstances, only those oocytes to be replaced may be inseminated. The purpose of this study was to develop a PGD protocol to diagnose first polar bodies (PBs) prior to Intracytoplasmatic Sperm Injection (ICSI) in order to inseminate only those oocytes found to be chromosomally normal. PB biopsy was performed 1 hour after ovum pick up, and after fixation, the PBs were analysed by FISH and the eggs inseminated by ICSI no later than 7 hours after retrieval. One third (33.3%) of the PBs were aneuploid. Fifty‐four normal and 12 non‐resolved oocytes were injected by ICSI, of which 65% became 2‐PN zygotes. Embryo transfer on day 2 was possible in all 10 patients (average maternal age 35.2±3.2, range 29–39 years), of which 6 became pregnant with 8 fetuses (28.6% or 8/28 transferred embryos). The results indicate that PB analysis of some common chromosome abnormalities is feasible within time limits imposed by ICSI insemination (6 hours or less). Copyright

Effect of embryo freezing on perinatal outcome after assisted reproduction techniques: lessons from the Latin American Registry of Assisted Reproduction

Embryo cryopreservation is an integral part of assisted reproduction techniques; it allows the sequential transfer of all embryos, thus diminishing the risk of multiple pregnancies and associated perinatal complications. To address concerns about the safety of this procedure, neonatal outcome after 43,070 fresh embryo transfers was compared with 12,068 frozen-thawed embryo transfers (FET). After adjusting for maternal age, gestational age, embryo development at time of transfer, number of babies born and gestational order, FET was not found to be associated with an increase in perinatal mortality (odds ratio [OR] 1.72, 95% confidence interval [CI] 0.81 to 3.62); preterm birth (OR 1.05, 95% CI 0.93 to 1.18); or extreme preterm birth (OR 0.82, 95% CI 0.64 to 1.06). Furthermore, after correcting for known confounding factors, FET was found to be associated with an increase in neonatal weight of 39.7 g (95% CI 1.54 to 64.10; P < 0.0001). Embryo cryopreservation was, therefore, not associated with an increase in the risk of poor perinatal outcome.

Outcome of assisted reproductive technology in overweight and obese women

Objective The main objective of this study was to assess the prevalence of overweight and obesity among patients undergoing assisted reproductive technology (ART) in Latin America and its consequences on treatment outcomes. Methods We used the Latin American Registry of ART to obtain womens age and body mass index (BMI), cancellation rate, number of oocytes retrieved and embryos transferred, clinical pregnancy, live birth and miscarriage rates from 107.313 patients undergoing autologous IVF and ICSI during four years; a multivariable analysis was performed to determine the effect of BMI on cancellation, oocytes retrieved, pregnancy, live birth and miscarriage, adjusting for age, number of embryos transferred and embryo developmental stage upon embryo transfer, when appropriate. Results The prevalence of overweight and obesity was 16.1% and 42.4%, respectively; correcting for age of female partner, overweight and obesity were associated to an increase in the odds of cancellation and to a lower mean number of oocytes retrieved; after adjusting for age, number of embryos transferred and stage of embryo development at transfer, we found that the BMI category was not associated to a change in the likelihoods of pregnancy, live birth and miscarriage. Conclusions The prevalence of obesity among women seeking ART in Latin America is surprisingly high; however, BMI does not influence the outcome of ART performed in these women.

Do women offered assisted reproduction technologies have a higher incidence of gynecologic cancer? A systematic review and meta-analysis

The last two decades have seen an increase in the number of women diagnosed with infertility. The consequent growth in the use of assisted reproductive technologies (ART) calls for the determination of its long-term effects, including the risk of cancer. Many studies have attempted to answer this question, albeit with contradictory results. This review aimed to assess whether assisted reproductive technologies are associated with an increased risk of gynecological cancer. A search for papers in the literature was carried out on MEDLINE, TRIP DATABASE and NICE, resulting in 11 studies enrolling 3,900,231 patients altogether. Of these, 118,320 were offered ART. The incidence of gynecological cancer in the group offered ART was 0.6%, while the incidence in the group not offered ART was 2.1%. Taking all the studies into consideration, women offered ART were not at greater risk of having gynecological cancer; instead, a protective association was found.

Male Factor Infertility Outcomes Using Magnetic Activated Cell Sorting in IntraCitoplasmatic Sperm Injection Cycles

Sperm DNA fragmentation can have negative consequences in clinical outcomes of couples undergoing Assisted Reproductive Technologies (ART). Sperm separation techniques are an important step in sperm selection for ART. The Magnetic Activated Cell Sorting (MACS) is a novel method that separates sperm by density gradient and molecular filtration to remove apoptotic sperm, which is associated to DNA damage. A decrease of DNA sperm fragmentation could improve ART outcomes. The main aim of this study was to assess the effect of MACS on fertilization, embryo development, implantation, clinical pregnancy and miscarriage rates, in couples undergoing intra cytoplasmic sperm injection (ICSI). Semen samples from 284 patients were divided in two groups; study group (n63) and control group (n = 221), analyzed by embryo transfer day (Day 3: ETD3 and day 5: ETD5) and male factor patients. Density gradients followed by MACS were used as sperm preparation method in the study group, while Swim up was the method used in the control group. Similar results were obtained between both groups for all parameters: fertilization rate of 77.18% versus 75.28%; blastulation rate of 46.66% versus 48.69%; implantation rate of 40.35% versus 35.52%; clinical pregnancy rate of 61.81% versus 59.31% and miscarriage rate of 2.94% versus 7.37%. However, statistical significant differences were found for implantation rate (study group 55.0% and control group 35.43%, p = 0.0138) in day 5 embryo transfers (ETD5). MACS technology does not improve general outcomes; however, it showed better results for ETD5. Further studies are required to identify real improvements in extended embryo culture in male infertility.

Embryo early cleavage, number of blastomeres and morphology at day three as factors to predict blastocyst development

Objective: the objective of this study is to evaluate markers of embryo quality, applied on day three of in vitro culture, in their capacity to predict whether embryos will reach blastocyst stage. Methods: a retrospective analysis of embryology database coming from 2772 zygotes obtained in 564 oocyte retrievals was performed; embryos were observed from day one to five: 23 to 26 hours after insemination all zygotes were evaluated to assess early cleavage, the number of blastomeres and embryo morphology were registered on day three and blastocyst formation was evaluated on day five; early first cleavage, number of blastomeres and embryo morphology at day three were correlated with embryo development until day five. Results: blastocyst formation rate was 36% in embryos with early cleavage and 19% in embryos without early

Does acupuncture the day of embryo transfer affect the clinical pregnancy rate? Systematic review and meta-analysis

The effects of acupuncture on IVF outcomes is still unknown. We carried out a systematic review and meta-analysis of RCT to determine whether acupuncture performed at the time of ET improves outcomes. We searched Medline and Embase from January 1990 to June 2017, for the following terms): (acupuncture; acupuncture therapy) and (reproductive techniques, assisted; in vitro fertilization; embryo transfer). We selected RCT that compared acupuncture with sham acupuncture or no treatment. We included only trials in which acupuncture involved the insertion of needles into traditional meridian points. We evaluated the methodological quality of the trials using the Cochrane risk of bias tool. The measure of treatment effect was the pooled odds ratio of achieving a clinical pregnancy, ongoing pregnancy, or live birth for women in the acupuncture group compared with women in the control group. For pooled data, summary test statistics were calculated using the Mantel-Haenszel method, using the Rev-Man software, version 5.1. We analyzed six studies, including 2,376. In all trials, there were no significant differences between the groups concerning the mean numbers of embryos transferred, the mean age of the women undergoing the procedure, diagnose and use of ICSI. Acupuncture performed the day of ET was associated with a reduced risk of clinical pregnancy (0.87, 95% confidence interval 0.77 to 0.98). The pooled rate difference was -0.06 (-0.12 to -0.01) for clinical pregnancy. None of the trials reported significant adverse effects of acupuncture.

There is no association between the presence of anti-thyroid antibodies and increased reproductive loss in pregnant women after ART: a systematic review and meta-analysis

Women submitted to ART treatments represent a select subgroup of individuals. Several studies have described the relationship between TAI and pregnancy outcomes as a result of ART, with contradictory results. The purpose of this systematic review was to determine the association between TAI and the risk of miscarriage in pregnancies resulting from ART. MEDLINE via PubMed, LILACS and Embase were searched for studies published in peer-reviewed journals from 1999 to 2017. The studies were summarized using the fixed effects model and the Petos method to calculate RR in order to flesh out the association between TAI and spontaneous abortion. Only four papers were included in this systematic review and meta-analysis. Thirty-one miscarriages were observed in 210 clinical pregnancies of women with antithyroid antibodies; and 158 miscarriages were seen in 1,371 pregnancies without antithyroid antibodies. The meta-analysis failed to find an association between TAI and higher risk of reproductive loss, RR=0.94 95% confidence interval: 0.71-1.24; p=0.879. In conclusion, the presence of antithyroid antibodies was not associated with increased reproductive loss in patients submitted to ART treatments. It is our opinion that the presence of antithyroid antibodies should be considered as a secondary biomarker of autoimmune disease, rather than an actual cause of miscarriage in patients undergoing ART. Due to the small amount of evidence on the matter, the determination of TAI before the initiation of ART should be limited to research contexts.