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 prospective and randomized trial comparing 225IU and 300 IU follitropin-α in a fixed-dose regimen for controlled ovarian stimulation / Estudo prospectivo e randomizado comparativo de 225 UI e 300 UI defolitropina-α em regime de dose fixa para estimulação ovariana controlada

Purpose: To compare the outcomes of 225 IU and 300 IU follitropin-α in a fi xed-dose regimen for controlled ovarian stimulation in women ≥35 years old. Material and methods: We studied 120 normo-ovulatory women ≥35 years old, undergoing IVF or ICSI cycles. After pituitary suppression, patients were randomly divided into two groups: G225 and G300. In G225 (n=60), ovarian stimulation was performed with a fixed daily dose of 225 IU of follitropin-α and in G300 (n=60), with a fixed daily dose of 300 IU, until hCG administration. The main outcomes were: the number of metaphase II oocytes retrieved, the percentage of MII oocytes, the cancellation rates, the number of days of stimulation and the fertilization rates. Data were analyzed statistically by the χ2 and Mann-Whitney tests, as p<0.05 was considered significant. Results: In G225, six cycles were cancelled (10%) and in G300, five cycles were cancelled(8.3%). The cancellation rates did not present statistical differences between groups (p>0.05). In G225, 301 oocytes were retrieved (5.02±1.32 per cycle); 261 were at MII stage. In G300, 338 o ocytes were collected (mean: 5.63±1.68 per cycle); 300 were at MII stage (p<0.05). The percentage of MII oocytes (86.7% in G225 versus 88.7% in G300), fertilization rate (69.7% in G225 versus 72.7% in G300), and the mean number of days of stimulation (9.7±0.6 in G225 versus 9.7±0.7 in G300) were not statistically different in both groups (p>0.05). Conclusions: We conclude that the dose of 225 IU r-FSH, rather than 300 IU, may be the dose of choice for ovarian stimulation in a fixed-dose regimen in this group of patients.

Objetivo: Comparar os resultados do uso de 250 UI e 300 UI de folitropina-α em regime de dose fixa em pacientes ≥35 anos de idade. Material e métodos: Foram estudadas 120 pacientes normo-ovulatórias submetidas a ciclos de fertilização in vitro ou injeção intracitoplasmática de espermatozoide. Apósa supressão hipofisária, as pacientes foram randomizadas nos grupos G225 e G300. No G225 (n=60), a estimulação ovariana foi realizada com 225 UI defolitropina-α e no G300 (n=60) com 300 UI, em regime de dose fixa até o dia do exame de hCG. Os resultados observados foram o número e porcentagem de oócitos em metáfase II coletados, taxa de cancelamento, número de dias de estímulo e taxa de fertilização. A análise estatística foi feita pelos testes χ2 e Mann-Whitney, considerando-se significante p<0,05. Resultados: No G225, houve seis ciclos cancelados (10%) e no G300 cinco (8,3%) (p>0.05). No G225, foram coletados 301 oócitos (5,02±1,32 por ciclo); 261 eram MII. No G300, foram coletados 338 oócitos (5,63±1,68 por ciclo), sendo 300 MII. A recuperação de oócitos MII (86,7% no G225 versus 88,7% no G300), as taxas de fertilização (69,7 versus 72,7%) e o número médio de dias de estimulação(9,7±0,6 versus 9,7±0,7) não foram estatisticamente diferentes entre os grupos (p>0,05). Conclusões: Concluímos que a dose de 225 UI de r-FSH podeser a dose de escolha para estimulação ovariana em regime de dose fixa nesse grupo de pacientes.

Eficácia do hCG em baixa dose para completar a estimulação ovariana iniciada com FSH recombinante / Efficacy of low dose hCG alone following recombinant FSH for controlled ovarian stimulation

O objetivo da presente comunicação é o de relatar os resultados de ciclos de FIV/ICSI com o emprego de FSH recombinante (r-FSH) durante todo o ciclo de estimulação e o uso de baixa dose de hCG isoladamente para completar a estimulação iniciada com r-FSH.

Baixa resposta ovariana: FIV convencional ou ICSI? / Poor ovarian response: conventional IVF or ICSI?

OBJETIVO: Avaliar as taxas de fertilização, clivagem e gravidez em pacientes submetidas à FIV convencional e à ICSI nos casos de má resposta ovariana. MATERIAL E MÉTODOS: Foi realizado estudo retrospectivo comparando-se os resultados de FIV convencional e ICSI em 55 pacientes com má resposta à estimulação ovariana. Foram comparados os seguintes parâmetros: idade, número de oócitos aspirados, taxa de fertilização, taxa de transferência, taxa de clivagem e taxa de gravidez por transferência. RESULTADOS: Não houve diferença estatisticamente significante entre os dois grupos em nenhum dos parâmetros avaliados (p maior que 0,05). CONCLUSÕES: Quando a estimulação ovariana e a aspiração folicular fornecerem no máximo 3 oócitos para o laboratório de reprodução assistida, a FIV convencional deve ser indicada, não havendo necessidade de se proceder à ICSI em tais situações

Testicular histopathological diagnosis as a predictive factor for retrieving spermatozoa for ICSI in non-obstructive azoospermic patients.

OBJECTIVE: Histological testicular pattern has a predictive role in the possibility of finding spermatozoa for ICSI in cases of non-obstructive azoospermia because some individuals could show residual spermatogenic sites in the testis. The aim of this study was to evaluate the sperm retrieval rate in each of the histopathological groups (hypospermatogenesis--Hypo, spermatogenic maturation arrest--MA, Sertoli cell only--SCO and testicular hyalinization) in patients assisted in our clinic. MATERIALS AND METHODS: Retrospective study from March 1997 to October 2002. We analyzed 14 patients with mean age of 34.3 +/- 0.7, with non-obstructive azoospermia. All patients were submitted to previous diagnostic biopsy (Bx) elsewhere and came to our institution for treatment. After an average of 12 months (8-20), they were submitted to a new Bx procedure to retrieve sperm. RESULTS: Previous diagnostic Bx showed the following histopathological results: 5 patients with MA, 4 with Hypo and 5 SCO. In the following Bx (for sperm retrieval) spermatozoa was found in 33% of the procedures in patients with MA, 50% in patients with Hypo and 40% of the procedures in patients with SCO. CONCLUSION: Previous diagnostic Bx can help in patient counseling concerning the result of sperm retrieval.

Testicular histopathological diagnosis as a predictive factor for retrieving spermatozoa for ICSI in non-obstructive azoospermic patients

OBJECTIVE: Histological testicular pattern has a predictive role in the possibility of finding spermatozoa for ICSI in cases of non-obstructive azoospermia because some individuals could show residual spermatogenic sites in the testis. The aim of this study was to evaluate the sperm retrieval rate in each of the histopathological groups (hypospermatogenesis-Hypo, spermatogenic maturation arrest-MA, Sertoli cell only-SCO and testicular hyalinization) in patients assisted in our clinic. MATERIALS AND METHODS: Retrospective study from March 1997 to October 2002. We analyzed 14 patients with mean age of 34.3 n 0.7, with non-obstructive azoospermia. All patients were submitted to previous diagnostic biopsy (Bx) elsewhere and came to our institution for treatment. After an average of 12 months (8 - 20), they were submitted to a new Bx procedure to retrieve sperm. RESULTS: Previous diagnostic Bx showed the following histopathological results: 5 patients with MA, 4 with Hypo and 5 SCO. In the following Bx (for sperm retrieval) spermatozoa was found in 33 percent of the procedures in patients with MA, 50 percent in patients with Hypo and 40 percent of the procedures in patients with SCO. CONCLUSION: Previous diagnostic Bx can help in patient counseling concerning the result of sperm retrieval.

High pregnancy rates with consecutive (two-step) embryo transfer in assistide reproductive technologies: preliminary results

Objetivo: O objetivo deste trabalho é verificar se a transferência embrionária dupla (consecutiva) pode proporcionar taxas de gravidez mais altas em técnicas de reprodução assistida. Material e métodos: Um total de 30 pacientes que tiveram 4 ou mais embriões para transferência no dia 3 após a coleta oocitária, foram submetidas a transferência embrionária dupla (consecutiva). Foram trasnferidos 3 embriões no dia 3 e um blastocisto no dia 5. Foram avaliadas as taxas de gravidez e implantação, determinadas pela visibilizaçào ecográfica de batimentos cardíacos fetais. Resultados: Dentre as 30 transferências embrionárias, houve 14 gestações (46,7%), sendo 4 (28,5%) únicas, 8 gemelares (57,1%) e 2 trigemelares (14,2%). Conclusões: Nossos resultados preliminares sugerem que a transferência consecutiva não apenas não afeta negativamente o processo de implantação embrionária, mas também relaciona-se com altas taxas de gravidez

Percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia

OBJECTIVES: Assessing the efficiency of repeated percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia, and also the possibility of cryopreservation of remaining material for future use in intracytoplasmic sperm injection (ICSI). METHOD: Retrospective study, in which 79 procedures of PESA were assessed in 58 patients (mean age = 45 years), whose partners had mean age of 34 years. Vasectomy was the most frequent cause of obstructive azoospermia (n = 46). RESULTS: Motile spermatozoa were obtained in 65 procedures (82 percent). PESA was twice repeated for 15 patients, 3 times for 5 patients, and 4 times for 1 patient. Spermatozoa were found in 13 (87 percent) patients in the second attempt, in 4 (80 percent) patients in the third attempt, and in the only patient that had accomplished 4 procedures. In 30 procedures (37 percent), we have obtained enough material for cryopreservation. In 12 among the 13 samples thawed (n = 13 patients), motile spermatozoa were found, and ICSI was accomplished. Four patients that did not use their samples requested the elimination of the material. Total rate of pregnancy per transference was 21/55 (38 percent). In 14 PESA procedures, it was not possible to find spermatozoa; in these cases, the patients opted for accomplishing the procedure of testicular sperm aspiration (TESA). CONCLUSION: PESA is an efficient and simple method of retrieving spermatozoa, allowing repeated procedures. Additionally, spermatozoa collected through PESA can be cryopreserved

Percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia.

OBJECTIVES: Assessing the efficiency of repeated percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia, and also the possibility of cryopreservation of remaining material for future use in intracytoplasmic sperm injection (ICSI). METHOD: Retrospective study, in which 79 procedures of PESA were assessed in 58 patients (mean age = 45 years), whose partners had mean age of 34 years. Vasectomy was the most frequent cause of obstructive azoospermia (n = 46). RESULTS: Motile spermatozoa were obtained in 65 procedures (82%). PESA was twice repeated for 15 patients, 3 times for 5 patients, and 4 times for 1 patient. Spermatozoa were found in 13 (87%) patients in the second attempt, in 4 (80%) patients in the third attempt, and in the only patient that had accomplished 4 procedures. In 30 procedures (37%), we have obtained enough material for cryopreservation. In 12 among the 13 samples thawed (n = 13 patients), motile spermatozoa were found, and ICSI was accomplished. Four patients that did not use their samples requested the elimination of the material. Total rate of pregnancy per transference was 21/55 (38%). In 14 PESA procedures, it was not possible to find spermatozoa; in these cases, the patients opted for accomplishing the procedure of testicular sperm aspiration (TESA). CONCLUSION: PESA is an efficient and simple method of retrieving spermatozoa, allowing repeated procedures. Additionally, spermatozoa collected through PESA can be cryopreserved.