Preimplantation genetic testing for aneuploidies does not increase success rates in fresh oocyte donation cycles: a paired cohort study.

PURPOSE: To determine whether in vitro fertilization cycles using fresh oocyte donations benefit from preimplantation genetic testing for aneuploidies. METHODS: A paired cohort study compared 44 fresh oocyte donation cycles with or without preimplantation genetic testing for aneuploidy (PGT-A). The sibling oocyte study analyzed fertilized oocytes, blastocyst development, and euploidy rate. Only frozen embryo transfers were performed. Pregnancy, implantation, biochemical pregnancy, miscarriage, stillbirth, live birth, and twin pregnancy rates were analyzed between groups. RESULTS: Fresh oocyte donation cycles between PGT-A and non-PGT-A groups were similar in all laboratory and clinical outcomes. A euploidy rate of 74.2% was observed in the PGT-A group. Although a slight trend was observed for implantation rate in the PGT-A group, it was not statistically significant. No difference was observed for live birth between groups. CONCLUSION: PGT-A associated with fresh oocyte donation cycles does not improve clinical outcomes and can be seen as over-treatment for patients.

Morphology-based selection from available euploid blastocysts induces male-skewed sex proportion in the offspring.

PURPOSE: To determine whether blastocyst morphology has an impact on sex proportion at pre-implantation and birth in PGT-A and non-PGT-A cycles. METHODS: A total of 1254 biopsied blastocysts from 466 PGT-A cycles were analyzed for sex proportion, day of biopsy, degree of expansion, inner cell mass (ICM), and trophectoderm (TE) morphology. From these, 197 frozen single embryo transfers (SET) were assessed for clinical outcomes and sex proportion of ongoing pregnancies and deliveries. In addition, we evaluated the day of vitrification/embryo transfer, degree of expansion, and TE morphology in a group of 229 births (217 cycles) from frozen or fresh transfers of non-biopsied blastocysts. RESULTS: Sex proportion was impacted by day of biopsy and TE morphology, but not by ICM morphology, in PGT-A cycles. Therefore, biopsy on day 5 and TE "A" shifted the sex proportion towards males. Interestingly, we noted that our morphology-based embryo selection for SET of euploid blastocysts has favored the choice for XY embryos, generating a 54.3% XY proportion at transfer and 56.1% XY proportion at ongoing pregnancy/delivery. Our models indicate a weaker association between blastocyst morphology parameters and sex proportion of babies in non-PGT-A cycles. CONCLUSION: Blastocyst features associated with a skewed sex proportion towards XY embryos, such as biopsy on day 5 and top quality TE, are also parameters used for selecting euploid embryos for SET. Therefore, our data suggest that morphology-based embryo selection represents a strong factor responsible for a skewed male sex proportion at birth in PGT-A cycles.

Platelet-rich plasma or blood-derived products to improve endometrial receptivity?

The use of platelet-rich plasma (PRP) to improve endometrial receptivity is gaining increasing attention in assisted reproduction technologies. The authors report that autologous PRP intrauterine administration improves pregnancy and birth rates, particularly in cases of patients presenting poor endometrial growth. Different groups of scientists proposed a similar approach years ago using whole blood-derived products also to improve endometrial receptivity. The important role played by cytokines and growth factors during embryo implantation has been well-known for a long time. These signaling molecules are present and released by blood cells during physiological, normal endometrial growth and implantation. Similar blood mediators are released from platelet granules upon a blood vessel injury. Methods described for PRP preparation for intrauterine administration are not precise, and they seem to be similar to those used to prepare peripheral blood-derived products. Thus, it is possible that when preparing PRP from whole blood, the final plasma product used as "PRP" contains platelets in addition to the important cytokines and growth factors released by the peripheral blood mononuclear cells present in the whole blood. Precise knowledge of the identity, concentration, and effects of the individual blood factors, their origin, whether platelets or blood mononuclear cells, will greatly contribute to improve and to make results obtained in fertility treatments more repeatable.

Platelet-rich plasma therapy and reproductive medicine.

Reports on clinical uses of platelet-rich plasma (PRP) have dramatically increased in the last decade. Indications for PRP therapy range from muscle and skeletal injuries to hair re-growth. More recently evidences have shown its positive effects in promoting endometrial and follicular growth and gestation in assisted reproduction cycles. We discuss the putative role of PRP on endometrial receptivity, with a brief history of its applications in research and clinical therapies. Despite its widespread uses in medicine, the mechanisms through which PRP exerts its regenerative effects are only postulated, not based on scientific data. There is an unmet need for advanced research to corroborate present findings in the clinical scenario.

Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container.

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.

Cytogenetics investigation in 151 Brazilian infertile male patients and genomic analysis in selected cases: experience of 14 years in a public genetic service.

OBJECTIVES: Male infertility accounts for approximately 30% of cases of reproductive failure. The characterization of genetic variants using cytogenomic techniques is essential for the adequate clinical management of these patients. We aimed to conduct a cytogenetic investigation of numerical and structural rearrangements and a genomic study of Y chromosome microdeletions/microduplications in infertile men derived from a single centre with over 14 years of experience. RESULTS: We evaluated 151 infertile men in a transversal study using peripheral blood karyotypes and 15 patients with normal karyotypes through genomic investigation by multiplex ligation-dependent probe amplification (MLPA) or polymerase chain reaction of sequence-tagged sites (PCR-STS) techniques. Out of the 151 patients evaluated by karyotype, 13 presented chromosomal abnormalities: two had numerical alterations, and 11 had structural chromosomal rearrangements. PCR-STS detected a BPY2 gene region and RBMY2DP pseudogene region microdeletion in one patient. MLPA analysis allowed the identification of one patient with CDY2B_1 and CDY2B_2 probe duplications (CDY2B and NLGN4Y genes) and one patient with BPY2_1, BPY2_2, and BPY2_4 probe duplications (PRY and RBMY1J genes).

A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles.

Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25ml plastic straw, vitrification block and fibreplug™). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9±18.4% when fibreplug and V16 (1.5M methanol+4.5M propylene glycol) solution were employed. When vitrified in V2 (1.5M methanol+5.5M Me2SO) the membrane integrity decreased to 42.0±21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance 2h post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes.

Substrates and supplements for hESCs: a critical review.

BACKGROUND: Different laboratories around the world have succeeded in establishing human embryonic stem cell (hESC) lines. However, culture conditions vary considerably among the protocols used and the vast majority of the lines at some stage of their creation have been in contact with an animal derived component. One of the main problems to be overcome for the generation of a clinical-grade hESC line is the choice of a substrate and medium that allows derivation and culture, where animal derived components are kept to a minimum or completely excluded. MATERIALS AND METHODS: The following review describes past and more recent achievements in the creation and culturing of hESC. It describes protocols, giving special attention to the matrices and supplements used for derivation, maintainance and cryostorage, considering whether they included defined, undefined and/or animal-derived components in their formulations. CONCLUSION: This information shall be useful for the creation and choice of new substrates and supplements for future research in the field of hESC for therapeutic purposes.

Balanced chromosomal rearrangement in a partner revealed after Preimplantation Genetic Testing for Aneuploidies (PGT-A).

In general population, it is estimated that 1/560 -1/1100 of the individuals are carriers of a balanced structural alteration and, in general, do not present an abnormal phenotype. For patients who have balanced rearrangements, a family planning alternative is to perform an In Vitro Fertilization (IVF) cycle with the embryonic analysis by Preimplantation Genetic Testing for Chromosomal Structural Rearrangements (PGT-SR). This test aims to reduce the time to obtain a healthy chromosomally pregnancy, to minimize the risk of miscarriage and a live birth with a chromosomopathy. The present work reports a case in which the couple had a history of implantation failure and biochemical pregnancy. They had not performed the karyotype exam to verify the parents' chromosomal content. After two embryo transfers without achieving pregnancy, the couple was directed to the Preimplantation Genetic Testing for Aneuploidies (PGT-A). The result presented in PGT-A in the couple's first cycle using the embryo selection technique showed recurrent segmental aneuploidies the trophectoderm biopsies. The couple was given genetic counselling, and they decided to investigate their karyotype, which showed a balanced chromosomal rearrangement in one of the parents. With this investigation and genetic counselling, it was possible to apply the correct embryonic analysis strategy, which contributed to a healthy pregnancy and birth with a living child.

Donor oocyte cycle characteristics and outcomes: factors potentially linked with successful endings.

OBJECTIVE: The use of donor oocytes in assisted reproduction has seen a significant rise worldwide in the last two decades. Postponement of motherhood and premature ovarian insufficiency are the main reasons for the increase in the number of in vitro fertilization cycles with donor oocytes. The present study aims to characterize donor oocyte cycles to analyze factors that may have an effect on live births and clinical pregnancy outcomes. METHODS: Data were obtained from a single Assisted Reproduction Center in southern Brazil. Recipient demographics (n=148 patients) and cycle characteristics (n=213 cycles; 50 patients did more than one IVF attempt) were analyzed. Statistical analysis was performed using chi-squared and t-test as appropriate. RESULTS: On average, recipients that reached gestation were significantly younger than the ones that did not. We also observed a significant positive effect of constant dose estrogen therapy on pregnancies. CONCLUSIONS: Patient age and response to estradiol therapy are important factors in the attainment of the best possible outcomes in cycles with donor oocytes.

The use of a metal container for vitrification of mouse ovaries, as a clinical grade model for human ovarian tissue cryopreservation, after different times and temperatures of transport.

PURPOSE: Cryopreservation of ovarian tissue is paramount for fertility preservation, with important clinical applications, especially for women suffering from an oncological condition. Several cryopreservation methodologies have been tried in search of better outcomes, especially in terms of primor-dial and primary follicles integrity post-cryopreservation. Vitrification has successfully been applied to ovarian tissue using different carriers for tissue exposure to the liquid nitrogen (LN2). METHODS: We developed an enclosed metal vessel, which has the advantage of a faster heat transfer, when in contact with LN2 avoiding at the same time, the direct contact with tissue. Additionally, we assessed the effect of different times and temperatures of transport between the collection of mouse ovaries and the beginning of cryopreservation, on follicular morphology after vitrification. RESULTS: Our results suggest that 37 °C and R.T. help to maintain normal primordial and primary follicle morphology for up to 4 hrs after collection and beginning of vitrification in a metal container. CONCLUSION: These data show that the metal container is an appropriate carrier for mouse ovary vitrification. The rate of morphologically normal primordial follicles up to 4 hrs.

Pronuclear and blastocyst morphology are associated age-dependently with embryo ploidy in in vitro fertilization cycles.

This retrospective study aimed to assess the relationship between standard markers of embryo morphology, maternal age and blastocyst ploidy determined by trophectoderm (TE) biopsy and Next-generation Sequencing (NGS). A total of 774 oocytes and embryos from 288 PGT-A cycles were scored for pronuclear, cleavage stage and blastocyst morphology. Pronuclear oocytes aligned between the nuclei and presenting equal number of nucleolus precursor bodies (NPBs) were designated Z1, oocytes showing equal number of NPBs, but not aligned, as Z2 while Z3 oocytes had an unequal number of NBPs between the nuclei or NPBs aligned in one nucleus and non-aligned in the other. Pronuclear oocytes with unequal-sized or non-aligned nuclei were designated Z4. Blastocysts were graded as BL1 (AA, AB or BA), BL2 (BB or CB) and BL3 (BC or CC) based on the combination of inner cell mass (ICM) and TE scores. Pronuclear and blastocyst morphology were correlated with aneuploidy in a < 40-year-old group (p < 0.01 and p < 0.05, respectively), but not in those ≥40 years. Interestingly, BL3 blastocysts classified as Z1 or Z3-Z4 on day-1 had different aneuploidy rates (BL3/Z1 = 46.7% vs. BL3/Z3-Z4 = 90.0%, p < 0.05). In summary, our data showed that pronuclear and blastocyst morphology are associated with blastocyst ploidy in younger patients. This may help embryo selection for embryo transfer and decision-making on which blastocysts should be biopsied in PGT-A cycles.

Morphological study on the effects of sample size on ovarian tissue vitrification.

OBJECTIVE: The present study aimed to assess the effects of ovarian cortex sample size on tissue morphological integrity after vitrification in a metal capsule. METHODS: Bovine ovarian tissue samples cut in large and small fragments (1x1x5 and 1x1x3 mm, respectively - 5 and 3 mm refer to length), vitrified in a metal capsule were fixed for histological analysis immediately after rewarming or after 48 hours culture. We assessed primordial, primary and secondary follicle morphology and stromal integrity. RESULTS: Primordial follicles showed the highest rates of normal morphology after rewarming and after 48 hours culture in both, small and large tissue fragments. Primary follicles presented a significant drop in normal morphology in large samples, after 48 hours in culture. Stromal integrity was well-preserved immediately after rewarming in small and large fragments but presented a significant drop in normal morphology in large samples, after 48 hours in culture. CONCLUSIONS: The ovarian reserve, represented by Primordial follicles, is well-preserved in small or large fragments, after vitrification and culture. However, the stromal components present better preservation after vitrification\rewarming, when tissue samples are cut in small fragments. Thus, small cortex samples should be preferred for ovarian tissue vitrification.

Fertilization outcome, embryo development and birth after unstimulated IVM.

BACKGROUND/PURPOSES: A common observation in oocyte in vitro maturation (IVM) cycles is poor embryo quality. However, no study was dedicated to assess zygote and early cleavage embryo quality in IVM cycles. The objective of this study is to analyze fertilization outcome, embryo development and the resulting pregnancy and births in unstimulated IVM cycles. METHODS: IVM oocytes were collected 36 h post hCG and matured in vitro for 28-30 h. All oocytes were inseminated by ICSI. Resulting zygotes and embryos were assessed on day-1, 2 and 3, when transfers were made. RESULTS: The overall oocyte maturation and fertilization rates were 63% and 62%, respectively. Abnormal fertilization rate was 27%. [corrected] Ninety five and 14.6% of the 2Pn zygotes reached the 2-cell and 8-cell stage at day-2 and day-3, respectively. Embryo quality assessment on day-3 at transfer revealed that only 9% of the embryos were of very good quality. Most embryos showed developmental delay. An average of 3.29 embryos were transferred per patient resulting in implantation and clinical gestation rates of 16% and 32%, respectively. Overall 14 healthy babies were born and there is one ongoing pregnancy. CONCLUSION: Results show a significant rate of abnormal fertilization and poor embryo quality after IVM, which is reflected in a higher than average number of embryos being transferred. However, pregnancy, implantation and birth rates are reasonably high and allow us to consider IVM a valuable approach for the treatment of infertility in PCO or PCOS patients.

First Baby Born in Brazil after Simultaneous Diagnosis through Non-Invasive and Conventional PGT-A.

Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) aiming to assess cell-free embryonic DNA in spent culture media is promising, especially because it might overcome the diminished rates of implantation caused by the inadequate performance of trophectoderm (TE) biopsy. Our center is part of the largest study to date assessing the concordance between conventional PGT-A and niPGT-A, and we report here the delivery of the first baby born in Brazil using niPGT-A. The parents of the baby were admitted to our center in 2018. They did not present history of infertility, and they were interested in using in vitro fertilization (IVF) and PGT-A in order to avoid congenital anomalies in the offspring. A total of 11 (3 day-5 and 8 day-6) expanded blastocysts were biopsied, and the spent culture media (culture from day-4 to day-6) from 8 day-6 blastocysts were collected for niPGT-A. Overall, 7 embryos yielded informative results for trophectoderm (TE) and media samples. Among the embryos with informative results, 5 presented concordant diagnosis between conventional PGT-A and niPGT-A, and 2 presented discordant diagnosis (1 false-positive and one false-negative). The Blastocyst 4, diagnosed as 46, XY by both niPGT-A and conventional PGT-A, was warmed up and transferred, resulting in the birth of a healthy 3.8 kg boy in February 2020. Based on our results and the recent literature, we believe that the safest current application of niPGT-A would be as a method of embryo selection for patients without an indication for conventional PGT-A. The approximate 80% of reliability of niPGT-A in the diagnosis of ploidy is superior to predictions provided by other non-invasive approaches like morphology and morphokinetics selection.

Abordagens para o teste genético pré-implantacional não-invasivo para aneuploidias (non-invasive preimplantation genetic testing for aneuploidies, niPGT-A, em inglês) com o objetivo de avaliar o DNA embrionário livre são promissoras, especialmente porque estas podem reverter as menores taxas de implantação causadas por inadequada biópsia de trofectoderma (TE). Nesse contexto, nosso centro é parte do maior estudo atual que avalia as taxas de concordância entre PGT-A convencional e niPGT-A, e relatamos aqui o nascimento do primeiro bebê brasileiro após niPGT-A. Os pais do bebê foram admitidos no nosso centro em 2018. Eles não apresentavam histórico de infertilidade, e estavam interessados em utilizar os tratamentos de fertilização in vitro (FIV) e PGT-A para evitar anomalias congênitas na progênie. Um total de 11 blastocistos expandidos (3 do dia-5 e 8 do dia-6) foram submetidos a biópsia, e os meios de cultivo condicionados (cultivo do dia-4 ao dia-6) de 8 blastocistos do dia-6 foram coletados para niPGT-A. No total, resultados informativos para as amostras de TE e dos meios foram obtidos para sete embriões. Entre os embriões com resultado informativo, 5 apresentaram diagnóstico concordante entre PGT-A convencional e niPGT-A, e 2 apresentaram diagnóstico discordante (1 falso positivo e 1 falso negativo). O Blastocisto 4, diagnosticado como 46, XY por ambos niPGT-A e PGT-A convencional, foi desvitrificado e transferido, o que resultou no nascimento de um menino saudável, que pesava 3,8 kg, em fevereiro de 2020. Com base em nossos resultados e literatura contemporânea, acreditamos que a aplicação atual mais segura do niPGT-A seria como método de seleção embrionária para pacientes sem indicação ao PGT-A convencional. A confiabilidade aproximada de 80% do niPGT-A para determinação da ploidia ainda é superior àquela obtida com abordagens não invasivas, como seleção morfológica ou morfocinética.

Comparison between two cryopreservation techniques of human ovarian cortex: morphological aspects and the heat shock response (HSR).

This study was tailored to compare the cryopreservation of the human ovarian cortex using closed metal container vitrification or the slow-freezing technique. Superficial ovarian cortical tissue biopsies were collected from 12 participants who underwent gynaecological videolaparoscopy. The fragmented samples were allocated to three experimental conditions: (a) fresh ovarian tissue, (b) slow-freezing, and (c) vitrification with a metal closed container. After thawing or rewarming, cellular morphological analyses were performed to determine tissue viability. The cellular response to thermal stress was measured by a putative increase in the immune quantification of the heat shock protein 70 kDa (heat shock protein 70 kDa response - HSR) after a heat challenge (2 h exposure at 42 °C). Both the total number of intact follicles and the frequency of primordial follicles were higher in fresh ovarian tissue than in the preserved samples, regardless of the technique employed. There was a trend towards an increase in the absolute number of intact follicles in the tissue preserved by vitrification. After cryopreservation, a higher HSR was obtained after slow-freezing. These results indicate that both cryopreservation techniques present advantages and may be used as alternatives to ovarian tissue cryopreservation.

Dichorionic twins and monochorionic triplets after the transfer of two blastocysts.

PURPOSE: To describe a unique case of MZ dichorionic twins and MZ monochorionic triplets in a quintuplet gestation after intracytoplasmatic sperm injection (ICSI) and blastocyst transfer. METHODS: Case report. A 24-year-old woman underwent ICSI and received two blastocysts transferred. A quintuplet gestation was established .Transvaginal ultrasonography was performed sequentially during early pregnancy. RESULTS: Three intrauterine gestational sacs were revealed at about 5th week. At the 7th week, five gestational sacs presenting heart beats were detected and a quintuplet pregnancy consisting of two monozygotic (MZ) dichorionic twins and three MZ monochorionic triplets was determined. At the 10th week, a single gestational sac with heart beats was detected. The prenatal course was uneventful. A healthy baby was born at 36th week. CONCLUSION: Few other reports have described the occurrence of a quintuplet gestation after the transfer of two blastocysts generated by ICSI. Our case is unique in that the two blastocysts underwent two different splitting processes, which occurred possibly at a similar time giving rise to MZ dichorionic twins and MZ monochorionic triplets.

Platelet-Rich plasma as an effective alternative approach for improving endometrial receptivity - a clinical retrospective study.

OBJECTIVE: The aim of the present case series was to describe our experience with the use of PRP on patients with refractory thin endometria. METHODS: This retrospective analysis included 24 IVF cycles in which patients presenting different infertility factors received intrauterine PRP infusion prior to embryo transfer. Outcomes of interest were: clinical and ongoing pregnancies, miscarriages, and births. RESULTS: 54% of the cycles in which PRP was employed resulted in ongoing gestation or birth; 12.5% of the cycles ended in miscarriages. CONCLUSION: Our data suggest that PRP improves intrauterine receptivity to embryo implantation, regardless of whether the endometrium reached the appropriate growth for embryo transfer.

Interaction of fibroblasts and induced pluripotent stem cells with poly(vinyl alcohol)-based hydrogel substrates.

Induced pluripotent stem cells (iPSCs) provide a promising means of creating custom-tailored cell lines for cellular therapies. Their application in regenerative medicine, however, depends on the possibility that the maintenance and differentiation of cells and organs occur under defined conditions. One major component of stem cell culture systems is the substrate, where the cells must attach and proliferate. The present study aimed to investigate the putative cytotoxic effects of poly(vinyl alcohol) (PVA)-based matrices on the in vitro culture of mouse fetal fibroblasts. In addition, the PVA-based hydrogels were used to determine the capacity of bovine induced pluripotent stem cells (biPSCs) to adhere and proliferate on synthetic substrates. Our results show that both cell types interacted with the substrate and presented proliferation during culture. The biPSCs formed new colonies when cell suspensions were placed onto the hydrogel surface for culture. These results may represent a new characterized xeno-free clinical grade culture system to be widely applied in cell-based therapies.