Prospective randomized study of D-Trp6-LHRH versus buserelin in long desensitization protocols for medically assisted conception cycles

In a prospective, controlled, randomized study where two differentagonists were used, we compared three different long desensitizationprotocols for induction of multiple follicular growth in medicallyassisted conception cycles. In protocol A, 30 patients wereinjected with buserelin twice a day for 15 days prior to ovarianstimulation until human chorionic gonadotrophin (HCG) administration.In protocol B, 30 patients were injected with a single doseof long acting Triptorelin (3.75mg) 15 days before the ovarianstimulation onset. In protocol C, 30 patients were injectedwith the long acting Triptorelin 4 weeks before ovarian stimulationfollowed by daily administration of 0.1 mg of the same agonistuntil HCG injection. There was no difference in the ovarianresponse to exogenous gonadotrophin stimulation, except forthe presence of premature luteinization in two patients in groupB. A significantly higher number of mature oocytes was collectedfrom patients with protocol A; however, the fertilization andcleavage rate demonstrated no significant difference among thethree groups of patients. The ongoing pregnancy rate and theimplanation rate per treatment cycle were very similar in thethree study groups. When the convenience, cost and side-effectsfor the patient are being considered, protocol B should be selectedas the first choice when the agonist is utilized for the purposeof inducing pituitary desensitization before and during ovarianstimulation.     

Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implantation rate

Human oocyte development was evaluated after a reduced timeexposure to spermatozoa in vitro. A total of 119 patients wereassigned to two study groups in a randomized prospective studyin which each patient‘s oocytes were exposed to spermatozoafor either 1 h (group 1 – 58 patients) or the standard16 h incubation period (group 2 – 61 patients). The fertilizationrate obtained in group 1 was higher than in group 2 (285/393,73%, and 272/410, 66% respectively), suggesting that the spermatozoa-oocyteinteraction occurs within 1 h. This was confirmed in a studyin vitro using fluorescently labelled spermatozoa and normaloocyte-cumulus complexes. Spermatozoa enter the cumulus complexwithin 15 min, traverse the cumulus layer within 3 h, and firstappear in the oocyte cortex at 4 h post-insemination. The incidenceof polyspermy was higher in oocytes exposed to spermatozoa for16 h (3%) than for 1 h (1%). There was no difference in thecleavage rate or morphological characteristics of embryos fromboth study groups. However, when evaluating the timing of embryodevelopment, group 1 generated a significantly higher percentageof four to five cell embryos when compared to group 2 (55 versus39%; P < 0.001), documented at 40 h post-insemination. Theimplantation and pregnancy rates for group 1 were 11 and 28%,while the corresponding rates for group 2 were 8 and 15%. Thissuggests that a reduced exposure of oocyte to spermatozoa favoursembryo viability, possibly due to a decrease in potential damagefrom sperm metabolic waste products.     

Guidelines for the appropriate use of genetic tests in infertile couples

Research on genetic causes of male and female infertility rapidly expanded in the last years, following the development of in vitro fertilising techniques. Genetic tests are now available to explore the cause of the infertility and assess the risk of a given couple to transmit its genetic characteristics. This allows at-risk couples to take an informed decision when electing for a medically assisted reproduction. It also allows the professionals to offer a prenatal diagnosis when appropriate. Thus, the genetic work-up of the infertile couple has become good practice for an appropriate diagnosis, treatment and prognostic assessment. The lack of national or international rules for the genetic approach to the infertile couple, prompted the Italian community of professionals in the field of reproductive medicine to join and set up guidelines for the genetic diagnosis of male and female infertility. The group of clinical and research experts is representative of 12 national scientific societies and was supported by external experts from four international societies. We examine the clinically relevant genetic causes of male and female infertility and suggest the category of patients for which each genetic test is recommended or optional, both for an accurate diagnosis and prior to ART.     

Effect of recombinant human gonadotrophins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro

The response of murine, bovine and human oocytes to pure recombinant preparations of human follicle stimulating hormone (rFSH) and luteinizing hormone (rLH) for meiotic maturation and subsequent developmental competence in vitro were examined in the present experiments. Maturation of immature bovine oocytes to the metaphase II stage was significantly increased by the addition of 1 IU/ml of rFSH in combination with either 1 IU/ml rLH or 10 IU/ml rLH. Similarly, embryonic development to the blastocyst stage was improved in bovine oocytes treated with a 1:10 combination of rFSH:rLH. However, no significant difference was observed in the number of inner cell mass or trophectoderm cells of the resulting blastocysts. Although the increased maturation to metaphase II was not significant, human embryonic developmental competence was improved by maturing oocytes in the presence of a 1:10 ratio of rFSH:rLH as only those oocytes exposed to a 1:10 ratio of rFSH: rLH during maturation showed normal cleavage patterns beyond day 2. In addition, 1 IU/ml rFSH and 1 IU/ml rLH increased the expression of oocyte proteins in human oocytes. The inclusion of recombinant gonadotrophins, either singly or in combination, had no significant effect on the maturation, fertilization or embryonic development of in-vitro matured mouse oocytes. These data provide support for the responsiveness of human and bovine oocytes to gonadotrophins in vitro and the need to consider variations in the relative concentrations for optimization of oocyte developmental competence.     

Incidence of Chromosomal Abnormalities from a Morphologically Normal Cohort of Embryos in Poor-Prognosis Patients

Purpose: Preimplantation genetic diagnosis of aneuploidy was performed on the embryos yielded by 70 poor-prognosis patients, with the aim of transferring those with a normal chromosomal complement, thus possibly increasing the chances of pregnancy. Methods: Multicolor fluorescence in situ hybridization (FISH) was applied for the simultaneous detection of chromosomes X, Y, 13, 16, 18, and 21. Inclusion criteria were (1) a maternal age of 36 years or older (n = 33), (2) three or more previous in vitro fertilization cycles (n = 20), and (3) an altered karyotype (n = 17). Results: A total of 412 embryos underwent FISH, resulting in 234 (57%) that were chromosomally abnormal. Euploid embryos were available for transfer in 59 patients, generating 19 pregnancies (32%), with an implantation rate of 19.9%. Conclusions: High rates of chromosomally abnormal embryos in poor-prognosis patients can determine repeated in vitro fertilization failures when embryo selection is performed on the basis of morphological criteria alone. Hence, the FISH analysis could represent the prevailing approach for the identification of embryos possessing full potential for developing to term.     

Substandard application of preimplantation genetic screening may interfere with its clinical success

The intent of this study was to evaluate a recent randomized clinical trial evaluating the effect of preimplantation genetic screening (PGS) that reports a negative effect on pregnancy outcome. This article reviews appropriate PGS techniques and how they differ from the trial in question. A closer look at the clinical trial in question reveals significant lack of expertise in biopsy, cell fixation, genetic analysis, and patient selection. At most, this trial demonstrates that in inexperienced hands, PGS can be detrimental. No other conclusions concerning the effect of PGS on pregnancy results can be drawn from the trial.     

Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed

OBJECTIVE: To verify whether advantages can derive from the implementation of preimplantation genetic diagnosis for aneuploidy in patients with a poor prognosis of full-term pregnancy, compared with conventional treatment procedures. DESIGN: A randomized, controlled study. SETTING: Reproductive Medicine Unit of the Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S): In a total of 262 stimulated cycles, women presented with the following poor-prognosis indications: maternal age of > or =36 years (n = 157), > or =3 previous IVF failures (n = 54), and an altered karyotype (n = 51). After giving consent, 127 patients underwent preimplantation genetic diagnosis for aneuploidy, whereas 135 controls underwent assisted zona hatching. INTERVENTION(S): Analysis of chromosomes XY, 13, 14, 15, 16, 18, 21, and 22 was carried out with the fluorescence in situ hybridization technique in a blastomere biopsied from day 3 embryos. Assisted zona hatching was performed on day 3 embryos from the control group. MAIN OUTCOME MEASURE(S): Embryo morphology and chromosomal status, number of transferred embryos, clinical pregnancies, implantation rates, and abortions. RESULT(S): In the study group, 717 embryos were analyzed by fluorescence in situ hybridization, and 60% were chromosomally abnormal. A mean of 2.3+/-0.9 euploid embryos were transferred in 99 cycles, resulting in 37 clinical pregnancies (37%) and a 22.5% ongoing implantation rate. In the control group, 126 cycles were performed with 3.2+/-1.3 embryos transferred, yielding 34 clinical pregnancies (27%) and a 10.2% ongoing implantation rate. CONCLUSION(S): The advantage of selecting embryos with a normal chromosome complement has an immediate impact on the ongoing implantation rate, especially in patients aged > or =38 years and carriers of an altered karyotype.     

Advantages of Day 4 Embryo Transfer in Patients Undergoing Preimplantation Genetic Diagnosis of Aneuploidy

Purpose: Following preimplantation genetic diagnosis of aneuploidy, embryo transfer was executed on day 4, with the aim of providing more time for expanding from six to nine the number of diagnosed chromosomes per single cell (Group 2; 45 cycles). The results obtained were compared to those derived from conventional day 3 transfer (Group 1; 71 cycles). Methods: For multicolor fluorescence in situ hybridization analysis, two panels of probes were used: the first, specific for chromosomes XY, 13, 16, 18, and 21, was tested in all patients (Groups 1 and 2); the second was implemented only in Group 2 patients for the detection of chromosomes 14, 15, and 22. Results: A total of 406 embryos underwent fluorescence in situ hybridization analysis in Group 1, and 236 in Group 2. Comparable percentages of both chromosomal abnormalities (61% and 62%) and pregnancy and implantation rates (36% and 24.5% in Group 1, 41% and 23.6% in Group 2) resulted, regardless of the higher mean age in Group 2. Conclusions: The diagnosis of the nine chromosomes which are most frequently associated with aneuploidy in humans could have an immediate impact on the rate of spontaneous abortions. Additional advantages are represented by the more accurate morphological evaluation of euploid embryos; the advanced compaction, which means that embryos are less exposed to damage during the transfer procedure; and the possibility of performing a reanalysis in cases where a fluorescence in situ hybridization diagnosis is not obtained.     

Over a decade of experience with preimplantation genetic diagnosis

The three respondents provide additional support for preimplantation genetic diagnosis (PGD) having the pivotal place it now has in prenatal genetic diagnosis: chromosomal abnormalities (e.g., unbalanced translocations), Mendelian disorders, and HLA typing for transfer of compatible, genetically normal, embryos. Transferring euploid embryos has decreased the clinical abortion rate and increased the implantation rate in assisted reproductive technologies (ART), but it has not necessarily improved the live-birth rate. Safer embryo biopsy, more extensive diagnostic efforts (i.e., microarray analysis), and more refined patient selection may be required before shifting from preselection of embryos based solely on morphological parameters to transfer of only aneuploidy-free embryos.     

Increased rate of aneuploid embryos in young women with previous aneuploid conceptions

OBJECTIVES: To determine whether a history of a previous aneuploid conception increases the rate of aneuploidy among women having preimplantation diagnosis (PGD). METHODS: Preimplantation embryos were tested for aneuploidy using FISH probes specific for chromosomes 13,15,16,17,18,21,22,X and Y.Using logistic regression to control for maternal age, we compared the rates of aneuploidy and other chromosome errors in 344 embryos from women having PGD because of a history of a previous aneuploid conception, 363 embryos from 42 women having PGD because of X-linked disorders and 1158 embryos from 135 women having PGD because of repeated in vitro fertilization failure. RESULTS: The frequency of aneuploidy differed significantly among patient groups for women younger than 35 (p = 0.003) but not for women older than 35. In women < 35, the rate of detected aneuploidy was 37.4% in the Aneuploid group, 20.9% in the X-linked group and 27.0% in the RIF group. (p = 0.0003 when the control groups are combined). The frequency of other chromosome abnormalities, as well as pregnancy and implantation rates, did not differ significantly among patient groups. CONCLUSIONS: This study suggests that a history of a trisomic pregnancy, whether or not it was a viable trisomy, is associated with an increased risk of another aneuploid conception at conception.