Josep M. Calafell

Evaluation of cytogenetic analysis for clinical preimplantation diagnosis

OBJECTIVE To evaluate the feasibility of using cytogenetic analysis in preimplantation diagnosis. DESIGN Two different biopsy protocols (chemical drilling and zona cutting) and two fixation methods were tested in a mouse model. Afterwards, the efficiency of obtaining chromosome preparations from untransferable human embryos depending on the method used to obtain the blastomeres (embryos biopsy or removal of the zona pellucida and blastomere disaggregation) was determined. The chances of obtaining chromosome preparations depending on the type of embryo (haploid, diploid, triploid, and apparently unfertilized) were also evaluated. RESULTS Results from the mouse model showed that chemical drilling yields better results than cutting in terms of metaphases per biopsied embryo and surviving rate after biopsy. In human embryos, biopsy of diploid embryos produced 46.6% chromosome preparations, while 29% were obtained after blastomere disaggregation and 20.4% when biopsying triploid embryos. CONCLUSIONS These results suggest that the disaggregating procedure and triploid embryos cannot be considered as good models to assess the feasibility of cytogenetic analysis in preimplantation diagnosis. Poor chromosome quality and loss during fixation are the main problems to use cytogenetics in preimplantation diagnosis; a combination of cytogenetics and other techniques is suggested in cases of balanced translocations.

Zona pellucida surface of immature and in vitro matured mouse oocytes: Analysis by scanning electron microscopy

PurposeThe aim of this work was to determine the morphology of the zona pellucida surface of immature and in vitro matured mouse oocytes by scanning electron microscopy. For this purpose two groups of immature oocytes (germinal vesicle group and metaphase I group) were studied either before or after in vitro maturation.ResultsBefore in vitro maturation, the germinal vesicle immature group showed mainly an unstructured zona pellucida surface with smooth cumulus cells. The metaphase I immature group showed a more structured zona pellucida with smooth or blebbing cumulus cells. After in vitro maturation, development of the zona pellucida toward a mature surface, related to the initial degree of oocyte maturity, was observed in both groups.ConclusionsThese observations show a correlation between the morphology of the zona pellucida surface and the degree of oocyte maturity; the in vitro maturation process can give rise to a proper development of this endowment when immature oocytes are used.

Early Hormonal Markers of Pregnancy Outcome After In Vitro Fertilization and Embryo Transfer

AbstractPurpose: To investigate the relative power of HCG, estradiol, and progesterone determinations in the prediction of pregnancy outcome after IVF. These prognostic hormonal factors were studied as single and combined predictors. Methods: Serum concentrations of β-HCG, progesterone, and estradiol were measured 12–13 days after embryo transfer (study point 1) and 7 days later (study point 2) in a series of 20 consecutive infertile patients having a first-trimester spontaneous clinical abortion after an IVF-embryo transfer cycle. As a control group (n=60), the next three IVF-embryo transfer cycles resulting in an ongoing pregnancy after each miscarried IVF cycle in our assisted reproduction program was used. The discrimination attained between the two study groups (ongoing pregnancies and miscarriages) was evaluated by logistic regression and receiver operating characteristic (ROC) curve analysis. Results: Mean hormone concentrations at study points 1 and 2 were higher in the ongoing pregnancy than in the abortion group. Regarding pregnancy outcome the percentage increment of HCG serum levels (≥1321%), with an accuracy (predictive value of pregnancy outcome) of 81.2% (sensitivity 98%, specificity 50%), had the best prognostic reliability but no significant differences were found when this parameter was compared with the predictive value of HCG concentration (≥72 IU/l) at study point 1 (diagnostic accuracy 80.5%; sensitivity 70%; specificity 80%). When ROC analysis was used, the best predictor of ongoing pregnancy according to the AUCROC was HCG concentration at study point 2 but again no significant differences were found when this parameter was compared with the predictive value of HCG serum levels at study point 1. A multiple marker strategy did not help distinguish viable from nonviable pregnancies. Conclusion: A single, early (days 12–13 after embryo transfer) HCG quantitative serum measurement in IVF cycles not only is diagnostic but also has good predictive value for pregnancy outcome.

The genetic risks of in vitro fertilization techniques: The use of an animal model

PurposeThe influence of some technical and biological parameters on the genetic characteristics of embryos derived from in vitro fertilization (IVF) techniques was studied.MethodUsing a murine model, we assessed the effect of gamete manipulation, gamete maturation stage, and maternal age on the chromosome complements of firstcleavage embryos.Results and ConclusionsWe found a positive correlation between some of these parameters and the incidence of the different chromosome abnormalities studied. Regarding aneuploidy, we observed an influence of maternal age, using both prepubertal and old females. Polyspermy showed a positive correlation with in vitro fertilization, the immaturity and overmaturity of the oocytes employed, and the use of prepubertal females. The appearance of diploid female complements was related to oocyte immaturity and prepubertal females, while diploid male complements were directly related to in vitro fertilization. Premature chromosome condensation (PCC) had a direct relationship with oocyte immaturity and in vitro maturation of the oocyte. Finally, structural abnormalities were associated with the process of sperm aging in vitro.

Pregnancy after orthotopic ovarian tissue transplantation using N-hexyl-2-cyanoacrylate

Cryopreservation of ovarian tissue in advance of cytotoxic therapies and later transplantation of the tissue is being performed increasingly often, and the total success rates in terms of pregnancy and delivery have been described in case series. Most pregnancies were achieved after orthotopic transplantation of tissue (in the peritoneum or the remaining ovary); however, treatment of the transplantation site during surgery is controversial. In this observational case-series study, we include four patients who underwent ovarian tissue transplantation between 2012 and 2016 by laparoscopy. Previously ovarian tissue was cryopreserved with slow freezing protocol prior to chemo- and/or radiotherapy. After cancer remission, the cryopreserved ovarian tissues were orthotopically re-transplanted in the ovarian medulla by laparoscopy, using N-hexyl-2-cyanoacrylate as an absorbable adhesion barrier. All patients regained ovarian function between 8 and 24 weeks after transplantation, as shown by follicle development and estrogen production. In patients 1 and 2 the ovarian function ended one year after transplantation. Patient 3 has regular menstrual cycles 2 years after the transplant and patient 4 currently has an ongoing spontaneous pregnancy. The use of N-hexyl-2 cyanoacrylate can facilitate the placement of ovarian pieces in orthotopic transplantation by laparoscopy without affecting the restoration and duration of ovarian activity.

Effect of anti-human sperm monoclonal antibodies on mouse in vitro fertilization

We employed anti-human sperm monoclonal antibodies to investigate how sperm membrane antigens are involved in gamete interactions. We have produced seven monoclonal antibodies specific for human sperm antigens, that showed reaction with mouse sperm by ELISA and by immunofluorescence. These antibodies did not react with zona pellucida or any other somatic human tissue. Some degree of toxicity was detected for oocytes at high antibody concentration and this was correlated with their inhibitory effect on fertilization. Unrelated to the degree of antigen expression or localization on sperm membrane, the antibodies showed several degrees of inhibition. The participation in sperm-zona pellucida interaction for every antigen could be evidenced by the impaired penetration of sperm caused by the presence of several concentrations of antibody. Thus, DAN-2, MOU-8 and VAC-4 inhibit mouse in vitro fertilization.