Cryopreservation of human oocytes is an important technique for the treatment of human infertility, as it deals successfully with legal, ethical, and moral issues related to embryo cryopreservation ...(Coticchio et al., Human Fertil (Camb) 4:152-157, 2001; Tucker et al., Eur J Obstet Gynecol Reprod Biol 113: 524-527, 2004) and maintains reproductive potential in women with diseases or conditions that may compromise reproductive capacity. Here, we describe an oocyte cryopreservation technique involving slow freezing-rapid thawing with differential sucrose concentration (0.2 M in the freezing stage-0.3 M in thawing stage) (Bianchi et al., Reprod Biomed Online. 14:64-71, 2007), describing the technique in detail, from the preparation of the solutions through to the performance of the technique and, finally, to a number of helpful hints for optimum results.
Purpose
Fine and balanced regulation of cell proliferation and apoptosis are key to achieve ovarian follicle development from the primordial to the preovulatory stage and therefore assure female ...reproductive function. While gonadotropins are the major and most recognized regulators of follicle cell growth and function, other factors, both systemic and local, play equally important roles. This work is aimed at evaluating the effects of thyroid hormones (THs) on human granulosa luteinized (hGL) viability.
Methods
Human GL cells derived from assisted reproduction treatments were exposed to T3 or T4. Cell viability was evaluated by MTT assay. Apoptosis was evaluated by the TUNEL assay and active caspase-3 staining.
StAR, CYP19A1,
Caspase
-
3, P53
and
BAX
mRNA were evaluated by real-time PCR. LC3-I/-II, AKT and pAKT were evaluated by western blot.
Results
T3 and T4 promoted cell viability in a dose-dependent modality and modulate
StAR
and
CYP19A1
expression. T3 and to a lesser extent T4 mitigated cell death induced by serum starvation by inhibition of caspase-3 activity and expression of
P53
and
BAX;
and attenuate cell death experimentally induced by C2-ceramide. Cell death derived from starvation appeared to be involved in autophagic processes, as the levels of autophagic markers (LC3-II/LC3-I ratio) decreased when starved cells were exposed to T3 and T4. This effect was associated with an increase in pAkt levels.
Conclusion
From the present study, THs emerge as potent anti-apoptotic agents in hGL cells. This effect is achieved by inhibiting the apoptosis signalling pathway of BAX and caspase-3, while maintaining active the PI3K/AKT pathway.
Abstract
Study question
Is the Vienna consensus appropriate to monitor IVF laboratory outcomes of treatments involving women of different age ranges?
Summary answer
Most IVF laboratory key ...performance indicators(KPIs) are reliably applicable irrespective of female age, while total good blastocyst rate should be adjusted according to female age
What is known already
The IVF laboratory is central to ART treatments. This demands methodical and precise monitoring of the performance. To assess laboratory efficiency, the Vienna consensus identified a set of relevant performance indicators(PIs) and KPIs applicable to a “reference population” defined by female age <40years, exclusion of PGT cases and use of own fresh oocytes and ejaculated sperm. It is known that women older than 39years – whose treatment outcome is affected by reduced oocyte quality–represent an increasingly large proportion of IVF patients. Thus, in this subgroup of patients the relevance of the Vienna indicators to treatments of remains to be demonstrated.
Study design, size, duration
Reported data concern a retrospective, single-center cohort analysis of 862 ART cycles carried out between January 2014 and May 2021. Inclusion criteria were indication for IVF/ICSI, blastocyst culture of all embryos formed in each cohort, use of own ejaculated spermatozoa (fresh or frozen), and complete cycles, i.e. those whose all embryos were transferred, cryopreserved or disposed of. Cancelled and PGT cycles were not included
Participants/materials, setting, methods
The overall population was divided into two groups according to female age: Vienna consensus (≤39 years) and older female age(≥40 years). We measured a selection of the Vienna performance indicators and KPIs, with a focus on measures relevant to embryo cleavage and blastocyst formation. Assessment of fertilization, cleavage and blastocyst rates was carried out. To assess more comprehensively blastocyst quality and quantity, we estimated the total usable blastocyst rate(TBUR). Finally, blastocyst cryosurvival rate was assessed.
Main results and the role of chance
No differences were observed in fertilization and embryo cleavage KPIs between the Vienna consensus and the older female age groups (standard IVF fertilization, 67.2 vs. 67.3; ICSI fertilization, 72.3 vs. 75.3; Day 2 development, 57.6% vs 58.7%; Day 3 development, 52.4% vs. 50.7 %, respectively). TBUR was lower in the older female age group (45.5% vs. 33.4% p < 0.001). This outcome decreased steadily with increasing female age. Clinical outcome significantly decreased with increasing female age. Implantation rate decreased from 34.3% in the Vienna consensus group to 16.9% in the older female age group (p<.001). Cumulative CPR showed a similar trend decreasing from 52.4% to 23.9% (p<.001). In the two populations, TBUR was further assessed after normalization of the number of retrieved oocytes. Rates were comparable in cycles with only few (1- 5 oocytes) collected oocytes (51.9% and 45.5%, p=ns). In cycles with 6-10, 11-15 and>15 collected oocytes TBUR was lower in older patient groups(p < 0.01). Univariate and multivariate logistic regression analysis showed female age as a factors independently associated with TBUR. Higher female age was associated with a reduced probability to achieve a TBUR greater than 40%.
Limitations, reasons for caution
The study design is retrospective and requires further refinement to control for factors that may impact clinical outcome
Wider implications of the findings
The study confirms the general validity of the Vienna Consensus but also indicate a need for fine-tuning in relation to factors intrinsic to gamete quality that can impact laboratory outcomes.
Trial registration number
Not applicable
Abstract
Study question
Does fertilization rate (FR) affect cumulative success rates in assisted reproduction cycles?
Summary answer
These data indicate a positive association between FR with CLBR ...suggesting the predictive clinical relevance of this parameter and its adoption as Key Performance Indicator(KPI).
What is known already
Numerous studies have aimed at characterizing outcome predictors. Maternal age is historically and correctly recognized as the single most important factor impacting on the clinical outcome of ART. More recently ovarian response has also gained interest in this respect. However, the quest for novel, more comprehensive predictive factors is not over; new relevant evidence is starting to emerge. FR is a noteworthy parameter because expressing a fundamental aspect of both oocyte and sperm developmental competence. In fact it has been adopted as a key performance indicator of the IVF laboratory, to assess laboratory, operator, and gamete competence.
Study design, size, duration
Reported data concern a retrospective cohort study carried out between 2015 to 2017 involving 7,968 couples undergoing 9,394 complete ICSI cycles, i.e. whose all embryos were transferred or disposed.All women aged between 18-42 years were included.We excluded from analysis: surgical sperm retrieval cases, cycles resulting in neither fresh or frozen–thawed embryo transfers,cycles in which live birth were not achieved, but with remaining cryopreserved embryos,cycles of PGT, cycle with fertilization failure and standard IVF cycles.
Participants/materials, setting, methods
The cohort was groupped according to fertilization rate intervals based on recommendations of the Vienna Consensus (<65% - Group 1; 65%-80% - Group 2; >80% - Group 3). Harnessing the large size of the original dataset, further cycle stratifications were carried out based on female age (<34, 35-38, 39-42 years) and number of oocytes retrieved (5-7, 8-10, >10 oocytes).
Main results and the role of chance
No significant difference in female age was observed between fertilization rate groups (p = 0.640). CLBR was progressively higher in relation fertilization rate in Groups 1, 2 and 3 (20.1%, 34.7%, 41.3%, P < 0.001, respectively). Number of recovered oocytes, embryo number per cycle, cumulative pregnancy rate followed the same trend (p < 0.001). The decrease in CLBR with increasing female age was significantly correlated with fertilization rate and CLBR in all three female age groups (P < 0.001). Finally, to further control for possible patient-specific confounding factors, maternal age, number of retrieved oocytes, percent of inseminated oocytes and fertilization rate were evaluated in a multivariate logistic regression analysis. From this assessment, fertilization rate emerged as a factor independently associated with cumulative live birth rate, to a degree equivalent or higher compared with the number or retrieved oocytes.
Limitations, reasons for caution
The study design is retrospective and requires further refinement to control for factors that may impact clinical outcome.
Wider implications of the findings
These data indicate a positive association of FR with CLBR, thereby suggesting that fertilization, in addition to representing an assay for gamete quality and laboratory performance,has an independent clinical significance.Irrespective of the number of retrieved oocytes and female age, we observed that, rates of FR are positively associated with CLBR.
Trial registration number
None
