PGT-HLA programmes for the cure of a sick sibling: clinical strategies for this challenging search.

The ultimate goal of a preimplantation genetic testing and human leukocyte antigen (PGT-HLA) matching programme is the birth of a healthy, HLA-compatible child for the treatment or cure of a sick sibling. Several authors have published successful cases of the births of children HLA-matched to siblings affected by different conditions and diseases. However, there are many reports of failed attempts. Couples seeking an HLA-matched sibling for their affected child look for positive outcomes in the shortest possible time. Nevertheless, there is no published consensus or guidelines with recommendations for these cases. Here, the authors aimed to analyse different approaches for these programmes, highlighting the most promising strategies for the families and fertility units. Furthermore, the authors mention a successful case of a PGT-HLA matching programme after a previous failed attempt following the strategies proposed. Which is the most cost-effective and time-efficient approach in a PGT-HLA matching programme?

Improving success rates by applying interventions in clinical practice and measuring their impact: A multicenter retrospective analysis of more than 240,000 cycles.

BACKGROUND: Systematic monitoring of key performance indicators (KPI) is an important component of quality management within the IVF laboratory and, as success of assisted reproduction depends on many variables, it is important to examine how each variable can be optimized to achieve the best possible outcome for patients. OBJECTIVE: To analyze how the design of a QMS impacts homogenization, safety, and efficacy in multiple fertility centers. Study Design Multicenter, retrospective cohort study with 188,251 patients who underwent 246,988 assisted reproductive treatments at 14 private centers belonging to IVI-RMA clinics between January 2005 and December 2019. Data were stratified by year, clinic, and patient group (standard patient cycles with no PGT-A, standard patients with PGT-A, and oocyte donation patients). Unadjusted and adjusted logistic regression models with other known predictors were made to analyze the impact and the interactions of policies. Main outcomes were determined per clinic and summarized per year as the median of the rates of the clinics; each clinic had the same weight independent of the number of cycles. RESULTS: Up to 188,251 patients were treated, for a total of 246,988 IVF cycles and 356,433 procedures. The introduction of standard operating procedures, trophectoderm biopsies, and blastocyst-stage transfers, coupled with an increased proportion of PGT-A cycles, led to improved outcomes while maximizing the number of single embryo transfers, driving a significant decrease in the number of multiple pregnancies while improving live birth rates. In terms of the live-birth rate per transfer, the interventions with greater impact over time in logistic regression analysis were 24-chromosome analysis and the introduction of benchtop incubators (odds ratio 1.92 [95% confidence interval 1.81 to 2.05]; p < 0.001). The odd ratios of the policies remained significant and very similar in the unadjusted and adjusted models. CONCLUSIONS: The greatest impact on live-birth rate per cycle was obtained with a cumulative effect of all policies, especially in egg donation patients. In patients without PGT-A changing embryo culture conditions and blastocyst stage transfer had the greatest impact; in patients with PGT-A, trophectoderm biopsy. Standardizing procedures was essential in reducing variability among clinics and implementing changes.

Diagnostic Accuracy Study Comparing Hysterosalpingo-Foam Sonography and Hysterosalpingography for Fallopian Tube Patency Assessment.

INTRODUCTION: Simplified ultrasound-based infertility protocols that appear to provide enough information to plan effective management have been described. Thus, the objective of this study is to compare the diagnostic accuracy of the hysterosalpingo-foam sonography (HyFoSy) in tubal patency testing with the traditional hysterosalpngography (HSG) for establishing a new diagnostic strategy in infertility. MATERIAL AND METHODS: Prospective observational diagnostic accuracy was performed in a private fertility clinic in which 106 women undergoing a preconceptionally visit were recruited. All of them had low risk for tubal disease, had performed an HSG and were negative for Chlamydia trachomatis antibody. Main outcome measures were tubal patency and pain grade. RESULTS: Evaluation of tubal patency by HyFoSy showed a total concordance with the results of the previous HSG in 72.6% (n = 77), and a total discordance for 4.7% (n = 6), with the inter-test agreement Kappa equal to 0.57, which means moderate concordance. Among the patients, 59.1% did not report pain during the procedure, while the remaining 48.1% indicated pain in different degrees; patients usually report less pain and only 6.6% described more pain with HyFoSy than with HSG (OR 6.57 (CI 95% 3.11-13.89)). Clinical outcomes after performing HyFoSy were not affected. CONCLUSIONS: HyFoSy is in concordance with HSG regarding tubal patency results and it is a less painful technique than HSG. HyFoSy is more economical and can be performed in an exam room only equipped with an ultrasound scanner. Based on these results, HyFoSy could be the first-choice diagnostic option to assess tubal patency in patients with low risk of tubal disease.

A picture of the covid-19 impact on IVIRMA fertility treatment clinics in Spain and Italy.

The emergence of the novel coronavirus infection that arose in Wuhan, China in December 2019 has resulted in an epidemic that has quickly expanded to become one of the most significant public health threats in recent times. Unfortunately, the disease has spread globally. On March 11th (2020) World Health Organization (WHO) declared Covid-19 a pandemic and has called governments to take urgent and aggressive action to change the course of the outbreak. Within the context of Assisted Reproduction, both reproductive medicine professionals and patients are also fighting against this unprecedented viral pandemic. In view of events, most of us had to make serious decisions, some of them with a lack of scientific evidence due to the circumstances and with the only objective of ensuring the safe care of our patients, reduce non-essential contacts and prevent possible maternal and fetal complications in future pregnancies. Pregnant women should not be considered at high risk for developing severe infection. Up to date, there are no reported deaths in pregnant women with Covid-19, while in the cases that have presented pneumonia because of Covid-19, the symptoms have been moderate and with a good prognosis in recovery.

Sam68 mediates leptin signaling and action in human granulosa cells: possible role in leptin resistance in PCOS.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is a complex metabolic disorder associated with ovulatory dysfunction, hyperandrogenism, obesity, and insulin resistance, that leads to subfertility. Sam68 is an RNA-binding protein with signaling functions that is ubiquitously expressed, including gonads. Sam68 is recruited to leptin signaling, mediating different leptin actions. OBJECTIVE: We aimed to investigate the role of Sam68 in leptin signaling, mediating the effect on aromatase expression in granulosa cells and the posible implication of Sam68 in the leptin resistance in PCOS. MATERIALS AND METHODS: Granulosa cells were from healthy donors (n = 25) and women with PCOS (n = 25), within the age range of 20 to 40 years, from Valencian Infertility Institute (IVI), Seville, Spain. Sam68 expression was inhibited by siRNA method and overexpressed by expression vector. Expression level was analysed by qPCR and immunoblot. Statistical significance was assessed by ANOVA followed by different post-hoc tests. A P value of <0.05 was considered statistically significant. RESULTS: We have found that leptin stimulation increases phosphorylation and expression level of Sam68 and aromatase in granulosa cells from normal donors. Downregulation of Sam68 expression resulted in a lower activation of MAPK and PI3K pathways in response to leptin, whereas overexpression of Sam68 increased leptin stimulation of signaling, enhancing aromatase expression. Granulosa cells from women with PCOS presented lower expression of Sam68 and were resistant to the leptin effect on aromatase expression. CONCLUSIONS: These results suggest the participation of Sam68 in leptin receptor signaling, mediating the leptin effect on aromatase expression in granulosa cells, and point to a new target in leptin resistance in PCOS.

Effect of Group Embryo Culture under Low-Oxygen Tension in Benchtop Incubators on Human Embryo Culture: Prospective, Randomized, Controlled Trial.

To evaluate the efficacy of group embryo culture under low-oxygen tension in benchtop incubators on human embryo development in vitro. The study was designed as a prospective, patient blind, randomized, controlled trial of a complex intervention. One hundred forty-eight women undergoing IVF were recruited in our fertility practice and randomized into two groups: intervention group (study culture strategy) or control group (control culture strategy). Intervention group embryos were cultured grouped under low-oxygen tension in benchtop incubators while control group embryos were cultured individually under atmospheric oxygen tension in large-box incubators. Using the study culture strategy, there were a significantly higher implantation rate (65.1% vs 49.2%; RR, 1.42; 95% CI, 1.17-1.73) and live birth delivery rate per embryo transfer (52.7% vs 39.5%; RR, 1.33; 95% CI, 1.02-1.75) with the first fresh embryo transfer. Cumulative implantation rate (56.7% vs 43.6%; RR, 1.30; 95% CI, 1.05-1.62) and cumulative live birth rate per embryo transfer (47.4% vs 36.2%; RR, 1.31; 95% CI, 1.01-1.69) were also statistically significantly increased in the study culture strategy. Human embryos exposed to our study culture condition strategy had statistically significant increased cumulative implantation rate and cumulative live birth rate per embryo transferred. Our findings suggest that this strategy specially favours poor quality embryos. Clinical Trial Registration Number: NCT01904006.

Altered expression of the kisspeptin/KISS1R and neurokinin B/NK3R systems in mural granulosa and cumulus cells of patients with polycystic ovarian syndrome.

PURPOSE: The neurokinin B (NKB)/NK3 receptor (NK3R) and kisspeptin (KISS1)/kisspeptin receptor (KISS1R), two systems essential for reproduction, are present in human granulosa cells (GCs) of healthy women and contribute to the control of fertility, at least partially, by acting on the gonads. However, little is known about the expression of these systems in GCs of women with polycystic ovarian syndrome (PCOS). The aim of this study was to analyze the expression of NKB/NK3R and KISS1/KISS1R in mural granulosa (MGCs) and cumulus cells (CCs) of PCOS women. METHODS: A cross-sectional study was performed in 46 healthy women and 43 PCOS women undergoing controlled ovarian stimulation. MGCs and CCs were collected from pre-ovulatory follicles after transvaginal ultrasound-guided oocyte retrieval and the expression of the genes encoding NKB (TAC3), NK3R (TACR3), KISS1, and its receptor (KISS1R) was analyzed using real-time quantitative RT-PCR. RESULTS: TAC3, TACR3, and KISS1 mRNA levels were decreased in MGCs and CCs of PCOS women. TAC3 positively correlated with KISS1 in MGCs of healthy women and TACR3 was positively associated with KISS1R in CCs from healthy women. These associations were not observed in PCOS women. CONCLUSION: The NKB/NK3R and KISS1/KISS1R systems are dysregulated in MGCs and CCs of PCOS women. The lower expression of these systems in GCs could contribute to the abnormal follicle development and defective ovulation that characterize the pathogenesis of PCOS.

Inter-laboratory agreement on embryo classification and clinical decision: Conventional morphological assessment vs. time lapse.

The aim of this study is to determine inter-laboratory variability on embryo assessment using time-lapse platform and conventional morphological assessment. This study compares the data obtained from a pilot study of external quality control (EQC) of time lapse, performed in 2014, with the classical EQC of the Spanish Society for the Study of Reproductive Biology (ASEBIR) performed in 2013 and 2014. In total, 24 laboratories (8 using EmbryoScope™, 15 using Primo Vision™ and one with both platforms) took part in the pilot study. The clinics that used EmbryoScope™ analysed 31 embryos and those using Primo Vision™ analysed 35. The classical EQC was implemented by 39 clinics, based on an analysis of 25 embryos per year. Both groups were required to evaluate various qualitative morphological variables (cell fragmentation, the presence of vacuoles, blastomere asymmetry and multinucleation), to classify the embryos in accordance with ASEBIR criteria and to stipulate the clinical decision taken. In the EQC time-lapse pilot study, the groups were asked to determine, as well as the above characteristics, the embryo development times, the number, opposition and size of pronuclei, the direct division of 1 into 3 cells and/or of 3 into 5 cells and false divisions. The degree of agreement was determined by calculating the intra-class correlation coefficients and the coefficient of variation for the quantitative variables and the Gwet index for the qualitative variables. For both EmbryoScope™ and Primo Vision™, two periods of greater inter-laboratory variability were observed in the times of embryo development events. One peak of variability was recorded among the laboratories addressing the first embryo events (extrusion of the second polar body and the appearance of pronuclei); the second peak took place between the times corresponding to the 8-cell and morula stages. In most of the qualitative variables analysed regarding embryo development, there was almost-perfect inter-laboratory agreement among conventional morphological assessment (CMA), EmbryoScope™ and Primo Vision™, except for false divisions, vacuoles and asymmetry (users of all methods) and multinucleation (users of Primo Vision™), where the degree of agreement was lower. The inter-laboratory agreement on embryo classification according to the ASEBIR criteria was moderate-substantial (Gwet 0.41-0.80) for the laboratories using CMA and EmbryoScope™, and fair-moderate (Gwet 0.21-0.60) for those using Primo Vision™. The inter-laboratory agreement for clinical decision was moderate (Gwet 0.41-0.60) on day 5 for CMA users and almost perfect (Gwet 0.81-1) for time-lapse users. In conclusion, time-lapse technology does not improve inter-laboratory agreement on embryo classification or the analysis of each morphological variable. Moreover, depending on the time-lapse platform used, inter-laboratory agreement may be lower than that obtained by CMA. However, inter-laboratory agreement on clinical decisions is improved with the use of time lapse, regardless of the platform used.

Expression of Tachykinins and Tachykinin Receptors and Interaction with Kisspeptin in Human Granulosa and Cumulus Cells.

The neurokinin B/NK3 receptor (NK3R) and kisspeptin/kisspeptin receptor (KISS1R), two systems which are essential for reproduction, are coexpressed in human mural granulosa (MGC) and cumulus cells (CCs). However, little is known about the presence of other members of the tachykinin family in the human ovary. In the present study, we analyzed the expression of substance P (SP), hemokinin-1 (HK-1), NK1 receptor (NK1R), and NK2 receptor (NK2R) in MGCs and CCs collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. RT-PCR, quantitative RT-PCR, immunocytochemistry, and Western blotting were used to investigate the patterns of expression of tachykinin and tachykinin receptor mRNAs and proteins and the possible interaction between the tachykinin family and kisspeptin. Intracellular free Ca(2+) levels ([Ca(2+)]i) in MGCs after exposure to SP or kisspeptin in the presence of SP were also measured. We found that SP, HK-1, the truncated NK1R isoform NK1R-Tr, and NK2R were all expressed in MGCs and CCs. NK1R-Tr mRNA and NK2R mRNA and protein levels were higher in MGCs than in CCs from the same patients. Treatment of cells with kisspeptin modulated the expression of HK-1, NK3R, and KISS1R mRNAs, whereas treatment with SP regulated kisspeptin mRNA levels and reduced the [Ca(2+)]i response produced by kisspeptin. These data demonstrate that the whole tachykinin system is expressed and acts in coordination with kisspeptin to regulate granulosa cell function in the human ovary.

The Metabolomic Profile of Spent Culture Media from Day-3 Human Embryos Cultured under Low Oxygen Tension.

Despite efforts made to improve the in vitro embryo culture conditions used during assisted reproduction procedures, human embryos must adapt to different in vitro oxygen concentrations and the new metabolic milieu provided by the diverse culture media used for such protocols. It has been shown that the embryo culture environment can affect not only cellular metabolism, but also gene expression in different species of mammalian embryos. Therefore we wanted to compare the metabolic footprint left by human cleavage-stage embryos under two types of oxygen atmospheric culture conditions (6% and 20% O2). The spent culture media from 39 transferred and implanted embryos from a total of 22 patients undergoing egg donation treatment was analyzed; 23 embryos came from 13 patients in the 6% oxygen concentration group, and 16 embryos from 9 patients were used in the 20% oxygen concentration group. The multivariate statistics we used in our analysis showed that human cleavage-stage embryos grown under both types of oxygen concentration left a similar metabolic fingerprint. We failed to observe any change in the net depletion or release of relevant analytes, such as glucose and especially fatty acids, by human cleavage-stage embryos under either type of culture condition. Therefore it seems that low oxygen tension during embryo culture does not alter the global metabolism of human cleavage-stage embryos.

Monozygotic twinning after assisted reproductive technologies: a case report of asymmetric development and incidence during 19 years in an international group of in vitro fertilization clinics.

OBJECTIVE: To describe a case of monozygotic twinning with asymmetric development following a single fresh embryo transfer as part of an intracytoplasmic sperm injection (ICSI) treatment. Secondarily, to report the incidence of monozygotic twinning at the IVI (Instituto Valenciano de Infertilidad) clinics. DESIGN: Case report. SETTING: Private fertility centers. PATIENT(S): A 33-year-old woman with a 2-year history of primary infertility. INTERVENTION(S): Controlled ovarian hyperstimulation and ICSI treatment with single-embryo transfer. MAIN OUTCOME MEASURE(S): Incidence of monozygotic twinning at the IVI clinics. RESULT(S): We report a twin pregnancy after a single-embryo transfer. Twins were dichorionic and diamniotic. One fetus had a 6-day delay in its growth compared with the other when observed by ultrasound. Two female infants were delivered, and despite presenting congenital diseases, they were successfully treated and evolved correctly. A subsequent DNA analysis confirmed that the infants were monozygotic. Furthermore, we estimated a monozygotic twinning rate of 1.17% at the IVI clinics, taking into account those cases in which two or more embryos with heart beats were observed by ultrasound scanning after single-embryo transfers. CONCLUSION(S): Ultrasound scans performed during pregnancy suggested a possible dizygotic origin of the twins, but DNA analysis performed after birth established that they were monozygotic. Genetic analysis is the only valid tool to confirm if like-sex dichorionic twins are monozygotic or dizygotic.

Elective single versus double embryo transfer: live birth outcome and patient acceptance in a prospective randomised trial.

The purpose of this study was to determine which strategy of embryo transfer has a better trade-off in live birth delivery rate versus multiple pregnancy considering patient acceptance: elective single embryo transfer (eSET) or elective double embryo transfer (eDET). In all, 199 women <38 years of age undergoing their first IVF treatment in a private centre were included in a prospective open-label randomised controlled trial. Patients were randomised into four groups: (1) eSET on Day 3; (2) eSET on Day 5; (3) eDET on Day 3; and (4) eDET on Day 5. Per patient, main analysis included acceptance of assigned group, as well as multiple and live birth delivery rates of the fresh cycle. Secondary analysis included the rates of subsequent cryotransfers and the theoretical cumulative success rate. Of 98 patients selected for eSET, 40% refused and preferred eDET. The live birth delivery rate after eDET was significantly higher after eDET versus eSET (65% vs 42%, respectively; odds ratio=1.6, 95% confidence interval 1.1-2.1). No multiple births were observed after eSET, compared with 35% after eDET. Although live birth delivery is higher with eDET, the increased risk of multiple births is avoided with eSET. Nearly half the patients refused eSET even after having been well informed about its benefits.

Influence of follicle rupture and uterine contractions on intrauterine insemination outcome: a new predictive model.

OBJECTIVE: To correlate the detection of follicle rupture and the number of uterine contractions per minute with the outcome of IUI and to build a predictive model for the outcome of IUI including these parameters. DESIGN: Retrospective cohort study. SETTING: Fertility clinic. PATIENT(S): We analyzed data from 610 women who underwent homologous or donor double IUI from 2005 to 2010 and whose data of uterine contractions or follicle rupture were recorded. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Live-birth rate. RESULT(S): Nine hundred seventy-nine IUI cycles were included. The detection of follicle rupture (odds ratio [OR], 1.98; 95% confidence interval [CI], 1.30-3.01) and the number of uterine contractions per minute (OR, 1.67; 95% CI, 1.02-2.74) assessed after the second insemination procedure of a double IUI were positively correlated with the live-birth rate. A multiple logistic regression model showed that sperm origin, maternal age, follicle count at hCG administration day, follicle rupture, and the number of uterine contractions observed after the second insemination procedure were significantly associated with the live-birth rate. CONCLUSION(S): Follicle rupture and uterine contractions are associated with the success of an IUI cycle. This may open new possibilities to improve the methodology of IUI.

Genetic damage following introduction of DNA in Phycomyces.

Introduction of plasmids in Phycomyces blakesleeanus caused extensive changes in the exogenous DNA and in the resident genome. Plasmids with a bacterial gene for geneticin resistance under a Phycomyces promoter were either injected into immature sporangia or incubated with spheroplasts. An improved method produced about one viable spheroplast per cell. Colonies resistant to geneticin were rare and only about 0.1% of their spores grew in the presence of geneticin. The transformation frequency was very low, < or =1 transformed colony per million spheroplasts or per microg DNA. Few nuclei in the transformants contained exogenous DNA, as shown by a selective procedure that sampled single nuclei from heterokaryons. The exogenous DNA was not integrated into the genome and no stable transformants were obtained. The plasmids were replicated in the recipient cells, but their DNA sequences were modified by deletions and rearrangements and the transformed phenotype was eventually lost. The spores developed in injected sporangia were often inviable; a genetic test showed that spore death was caused by impaired nuclear proliferation and induction of lethal mutations. About one-fourth of the viable spores from injected sporangia formed abnormal colonies with obvious changes in shape, texture, or color. The abnormalities that could be investigated were due to dominant mutations. The results indicate that incoming DNA is not only attacked, but signals a situation of stress that leads to increased mutation and nuclear and cellular death.