Relationship between age and blastocyst chromosomal ploidy analyzed by noninvasive preimplantation genetic testing for aneuploidies (niPGT-A).

OBJECTIVE: To assess the relationship between human blastocyst chromosomal ploidy established by niPGT-A and increasing age. METHODS: This is a prospective multicenter study carried out by ten assisted reproduction centers after their embryologists acquired training and validated their results with the previous use of niPGT-A. A total of 94 couples with indication for niPGT-A due to increase maternal age, male factor, repeated implantation failures, recurrent abortion or because they requested niPGT-A were included in this study. The couples had no karyotype abnormalities. After ICSI, the embryos were cultured until blastocyst stage using one or two step culture systems, single or sequential media respectively, at 37°C in an atmosphere of 6-7% CO2 and 5-20% O2 incubators. On day 3, we re-evaluated cleavage embryos to complete cumulus cells removal. The embryos were then cultured in individual well, with 20µl of medium under oil until they reached blastocyst stage. The blastocysts were vitrified and stored in liquid nitrogen. After that, the spent blastocyst culture medium (20µl) was transferred to a PCR tube and sent for analysis in the genetic laboratory, where it was stored at -80°C until sequencing. A total of 243 samples of spent blastocyst culture medium were collected on the 5th/6th day. Cell-free DNA secreted on culture medium was amplified using NICS Sample Preparation Kit (Yikon Genomics), based on the MALBAC technology. After whole genome amplification, the DNA was measured using a Qubit 2.0 fluorometer and subjected to next generation sequencing (NGS) using Illumina MiSeq® platform. The data were analyzed using the ChromGo® software (Yikon Genomics). RESULTS: The mean age of the patients was 38±4.08 years with an interval of 20-44 years. The euploid was diagnosed in 36.4% (80/220) of cases, aneuploidy in 31.3% (69/220), and mosaicism in 32.3% (71/220; with ≥60% aneuploidy) of blastocysts. Mosaic values ranged from 29.8% to 33.8% in different age groups. Individually, the most frequent chromosomal abnormality was XXY (Klinefelter Syndrome) occurring in 18 cases, followed by chromosome 21 (trisomy/monosomy) in 8 cases. The niPGT-A data showed a ≥60% incidence of aneuploid cells in all cases of chromosomal mosaicism (n=71). CONCLUSION: A high degree of mosaicism with aneuploidy cells was detected, and some hypotheses were suggested for this data (niPGT-A sensitivity in detecting the self-correction of chromosomal abnormalities phenomenon). However, it did not vary remarkably with age. On the other hand, euploidy levels had a negative correlation with age and aneuploidy levels had a positive relationship. This is the first report in the literature to relate chromosomal ploidy in blastocysts using niPGT-A and increasing patient age.

A comparison between a new vitrification protocol and the slow freezing method in the cryopreservation of prepubertal testicular tissue.

OBJECTIVE: This study aimed to compare a new vitrification protocol with reduced cryoprotectant exposure to the slow freezing method in the cryopreservation of prepubertal rat testicular tissue. METHODS: Five sexually immature male Wistar rats were submitted to bilateral orchiectomy. Tissue samples from each testicle were fragmented into small pieces and randomly assigned to three groups: Group A, fresh tissue (control); Group B, slow programmable freezing (SPF); and Group C (vitrification). Frozen/thawed, vitrified/warmed, and fresh testicular tissue were histologically compared. A pathologist blinded to the procedures assessed the morphology (cell differentiation, nuclei, and epithelium) of 10 seminiferous tubules from each testicle (100 tubules per Group). RESULTS: Sertoli and spermatogonial stem cells were easily differentiated, and the nucleoli were easily viewed in the tubules assessed in all three groups. Small alterations in tissue architecture were observed in the control group as a result of tissue handling. Moderate alterations of the epithelium with the formation of small gaps and cell detachment from the basement membrane were observed in 28% of the frozen and 9% of the vitrified tubules. Condensed nuclei involving a small proportion of cells were observed in six and three tubules of the frozen and vitrified group, respectively. Despite the alterations, 97% of the frozen and 99% of the vitrified tubules were considered well preserved. CONCLUSIONS: The findings indicate that the vitrification protocol tested in this study adequately preserved the morphological integrity of prepubertal testicular tissue in a rat model. Further studies are required to confirm testicular tissue function after grafting.

Viability of homologous and heterologous subcutaneous transplantation of fresh germ cells in rabbits.

OBJECTIVE: This study aimed to compare heterologous to homologous transplantation of fresh ovarian germ cells in rabbits. METHODS: Twelve female white New Zealand rabbits (Oryctolagus cuniculus) were randomly numbered and submitted to bilateral oophorectomies. The ovaries from the six odd-numbered rabbits were dissected and cortical germinal tissue was digested in collagenase type 1 to obtain six solutions containing stromal and germ cells, which were injected in the abdominal region of the odd-numbered rabbits themselves (homologous transplantation) and of the even-numbered rabbits (heterologous transplantation) off immunosuppression. Sixty days after transplantation, the tissue around the transplanted region was excised, processed and sent to histological analysis with hematoxylin-eosin staining and Bcl-2 immunohistochemistry to verify the presence and viability of the transplanted cells. RESULTS: The analyzed specimens contained ovarian stroma, while follicular cells were found in 66.6% of the homologous and in 60% of the heterologous transplant specimens. Mild inflammatory reaction was observed in all heterologous specimens, and in only one (16.7%) of the homologous specimens. However, this inflammatory reaction was not so intense as to cause the death of the implanted cells. Except for the specimens from rabbits 7 and 8, all specimens were stained for Bcl-2, indicating that most of them were viable. CONCLUSIONS: The results of this study supported the viability of heterologous transplantation of fresh ovarian germ cells. However, more studies are required to further our understanding and improve the germ cell separation technique.

Vitrification of Human Oocytes and its Contribution to In Vitro Fertilization Programs.

OBJETICVE: To study the cumulative pregnancy outcome, particularly in terms of live births, with the consecutive transfer of embryos from fresh and vitrified/warmed oocytes to infertile patients in a routine infertility program. METHODS: Patients were initially submitted to in vitro fertilization embryo transfer with fresh embryos, while surplus oocytes were vitrified with the Vitri-Ingá method. Patients who did not succeed to carry their gestation to term underwent a new cycle with embryos from their own warmed oocytes. Some of the patients participating in the first warming cycle, who still possessed surplus oocytes, underwent a second warming cycle. Clinical and pregnancy outcomes obtained with fresh and warming cycles were compared using the chi-square test at a level of significance of 5%. RESULTS: Of the 211 participating patients, 97 (46%) got pregnant with fresh embryo transfer, and 69 (32.7%) carried their pregnancies to term. Of the patients participating in the first and second warming cycles, 32/100 (32%) and 6/20 (30.0%) resulted in live births, respectively. Thus, of the 211 participating patients, 107 carried their pregnancies to term, representing a cumulative live birth rate of 50.7%. No statistically significant differences between the use fresh and vitrified oocytes were found for any of the variables studied. CONCLUSIONS: Oocyte vitrification offered the possibility of gestation in more than one attempt after just one controlled hyperstimulation. Apart from alleviating the financial burden on patients, vitrification of oocytes may result in a feasible solution for the problems generated by abandoned frozen embryos.

The First Ovarian Tissue Transplant between Monozygotic Twin Sisters Discordant for Ovarian function in Latin America.

Ovarian tissue transplant is an alternative to the cryopreservation of oocytes and embryos for the recovery of fertility and natural hormonal activity. The objective of this paper is to report on the first fresh ovarian tissue transplant between monozygotic twin sisters discordant for ovarian function, using the subcortical implant technique of ovarian tissue fragments, to take place in Latin America. A strip representing approximately a quarter of the cortical tissue was removed from the right ovary of the donor sister, cleaned, cut into small fragments and sent to adjacent room, where the receptor sister was concomitantly being prepared to receive the tissue graft. The ovarian fragments were placed under the cortical tissue onto a vascularized bed of the right ovary of the receptor sister. From 90 days postoperatively, the menstrual cycles of the receptor patient became regular with increased flow and longer periods, demonstrating normal hormonal activity and improved endometrial development. Attempts at spontaneous pregnancy, and the recovery of an oocyte followed by fertilization have not yet been successful. However, the ovarian tissue transplant between monozygotic sisters reported here clearly highlights the potential of the technique as a therapeutic option for the preservation of fertility.

Laboratório para fertilizaçäo in vitro / Laboratory setup for in vitro-fertilization

A instalaçäo de um laboratório de fertilizaçäo in vitro inicia-se pelo planejamento da equipe que o comandará, para após escolher o protocolo de induçäo da ovulaçäo, como colher os oócitos, como manusear os oócitos e embriöes, como preparar os espermatozóides e inseminar e como transferir os embriöes. Apesar da situaçäo econômica atual e dos altos investimentos, a fertilizaçäo in vitro já é factível a custos ao alcance de grande parte da populaçäo, principalmente com a entrada no mercado de meios de cultura prontos para uso e de fabricantes brasileiros de materiais de consumo.

Instalaçäo de banco de sêmen / Semen bank installation

Para a instalaçäo de um banco de sêmen e necessário planejar o cadastramento das amostras, critérios de avaliaçäo dos doadores e do sêmen, colheita e classificaçäo do material, escolha do meio crioprotetor, armazenamento e recuperaçäo dos espermatozóides após descongelamento. Os espermatozóides armazenados säo utilizados para inseminaçöes ou fertilizaçöes, homóloga ou heteróloga, aumentando o sucesso da clínica de reprodutpo.

Síndrome de hiperestimulaçäo ovariana: relato de um caso / Ovarian hyperestimulation syndrome: a case report

The authors report a case of ovarian hyperstimulation syndrome in ovulation induction with clomifene citrate, HMG and HCG to in vitro fertilization. There was total retum with an albumina I.V. and emphasize the estradiol in a series of dosages and the folicular development by ultrasound