Reference standards for follicular density in ovarian cortex from birth to sexual maturity.

RESEARCH QUESTION: Are age-normalized reference values for human ovarian cortical follicular density adequate for tissue quality control in fertility preservation? DESIGN: Published quantitative data on the number of follicles in samples without known ovarian pathology were converted into cortical densities to create reference values. Next, a sample cohort of 126 girls (age 1-24 years, mean ± SD 11 ± 6) with cancer, severe haematological disease or Turner syndrome were used to calculate Z-scores for cortical follicular density based on the reference values. RESULTS: No difference was observed between Z-scores in samples from untreated patients (0.3 ± 3.5, n = 30) and patients treated with (0.5 ± 2.9, n = 48) and without (0.1 ± 1.3, n = 6) alkylating chemotherapy. Z-scores were not correlated with increasing cumulative exposure to cytostatics. Nevertheless, Z-scores in young treated patients (0-2 years -2.1 ± 3.1, n = 10, P = 0.04) were significantly lower than Z-scores in older treated patients (11-19 years, 2 ± 1.9, n = 15). Samples from patients with Turner syndrome differed significantly from samples from untreated patients (-5.2 ± 5.1, n = 24, P = 0.003), and a Z-score of -1.7 was identified as a cut-off showing good diagnostic value for identification of patients with Turner syndrome with reduced ovarian reserve. When this cut-off was applied to other patients, analysis showed that those with indications for reduced ovarian reserve (n = 15) were significantly younger (5.9 ± 4.2 versus 10.7 ± 5.9 years, P = 0.004) and, when untreated, more often had non-malignant haematologic diseases compared with those with normal ovarian reserve (n = 24, 100% versus 19%, P = 0.009). CONCLUSION: Z-scores allow the estimation of genetic- and treatment-related effects on follicular density in cortical tissue from young patients stored for fertility preservation. Understanding the quality of cryopreserved tissue facilitates its use during patient counselling. More research is needed regarding the cytostatic effects found in this study.

A double-blind randomized controlled trial investigating a time-lapse algorithm for selecting Day 5 blastocysts for transfer.

STUDY QUESTION: Can use of a commercially available time-lapse algorithm for Day 5 blastocyst selection improve pregnancy rates compared with morphology alone? SUMMARY ANSWER: The use of a time-lapse selection model to choose blastocysts for fresh single embryo transfer on Day 5 did not improve ongoing pregnancy rate compared to morphology alone. WHAT IS KNOWN ALREADY: Evidence from time-lapse monitoring suggests correlations between timing of key developmental events and embryo viability. No good quality evidence exists to support improved pregnancy rates following time-lapse selection. STUDY DESIGN, SIZE, DURATION: A prospective multicenter randomized controlled trial including 776 randomized patients was performed between 2018 and 2021. Patients with at least two good quality blastocysts on Day 5 were allocated by a computer randomization program in a proportion of 1:1 into either the control group, whereby single blastocysts were selected for transfer by morphology alone, or the intervention group whereby final selection was decided by a commercially available time-lapse model. The embryologists at the time of blastocyst morphological scoring were blinded to which study group the patients would be randomized, and the physician and patients were blind to which group they were allocated until after the primary outcome was known. The primary outcome was number of ongoing pregnancies in the two groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: From 10 Nordic IVF clinics, 776 patients with a minimum of two good quality blastocysts on Day 5 (D5) were randomized into one of the two study groups. A commercial time-lapse model decided the final selection of blastocysts for 387 patients in the intervention (time-lapse) group, and blastocysts with the highest morphological score were transferred for 389 patients in the control group. Only single embryo transfers in fresh cycles were performed. MAIN RESULTS AND THE ROLE OF CHANCE: In the full analysis set, the ongoing pregnancy rate for the time-lapse group was 47.4% (175/369) and 48.1% (181/376) in the control group. No statistically significant difference was found between the two groups: mean difference -0.7% (95% CI -8.2, 6.7, P = 0.90). Pregnancy rate (60.2% versus 59.0%, mean difference 1.1%, 95% CI -6.2, 8.4, P = 0.81) and early pregnancy loss (21.2% versus 18.5%, mean difference 2.7%, 95% CI -5.2, 10.6, P = 0.55) were the same for the time-lapse and the control group. Subgroup analyses showed that patient and treatment characteristics did not significantly affect the commercial time-lapse model D5 performance. In the time-lapse group, the choice of best blastocyst changed on 42% of occasions (154/369, 95% CI 36.9, 47.2) after the algorithm was applied, and this rate was similar for most treatment clinics. LIMITATIONS, REASONS FOR CAUTION: During 2020, the patient recruitment rate slowed down at participating clinics owing to coronavirus disease-19 restrictions, so the target sample size was not achieved as planned and it was decided to stop the trial prematurely. The study only investigated embryo selection at the blastocyst stage on D5 in fresh IVF transfer cycles. In addition, only blastocysts of good morphological quality were considered for transfer, limiting the number of embryos for selection in both groups: also, it could be argued that this manual preselection of blastocysts limits the theoretical selection power of time-lapse, as well as restricting the results mainly to a good prognosis patient group. Most patients were aimed for blastocyst stage transfer when a minimum of five zygotes were available for extended culture. Finally, the primary clinical outcome evaluated was pregnancy to only 6-8 weeks. WIDER IMPLICATIONS OF THE FINDINGS: The study suggests that time-lapse selection with a commercially available time-lapse model does not increase chance of ongoing pregnancy after single blastocyst transfer on Day 5 compared to morphology alone. STUDY FUNDING/COMPETING INTEREST(S): The study was financed by a grant from the Swedish state under the ALF-agreement between the Swedish government and the county councils (ALFGBG-723141). Vitrolife supported the study with embryo culture dishes and culture media. During the study period, T.H. changed his employment from Livio AB to Vitrolife AB. All other authors have no conflicts of interests to disclose. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov registration number NCT03445923. TRIAL REGISTRATION DATE: 26 February 2018. DATE OF FIRST PATIENT'S ENROLMENT: 11 June 2018.

Ovarian response is affected by a specific histidine-rich glycoprotein polymorphism: a preliminary study.

Genetic polymorphisms involved in angiogenesis, apoptosis and chemokine signalling are associated with varying ovarian response and oocyte quality. The protein, histidine-rich glycoprotein (HRG), is involved in these processes, but its effect on ovarian response in IVF has not been previously studied. A single nucleotide polymorphism (SNP) in the HRG gene (C633T) seems to affect pregnancy results in IVF. Women with the C/C genotype had higher pregnancy rates, C/T had moderate rates and none of those in the T/T group conceived. The aim of this study was to investigate if the HRG C633T SNP affects ovarian response. The HRG C633T SNP genotype of 67 women with unexplained infertility undergoing IVF was analysed and related to medical data. The T/T genotype obtained fewer oocytes, including mature oocytes, despite higher dosages of FSH administered. Additionally, the highest proportion of women who had exclusively poor-quality embryos was in the T/T group. No differences in demographic factors known to affect these parameters were found. The results suggest that the HRG C633T SNP influences ovarian response. Further studies of this SNP may increase knowledge about the biological processes involved in oocyte development and, furthermore, improve predicted ovarian response and fertilization.

Polyethylene glycated leukemia inhibitory factor antagonist inhibits human blastocyst implantation and triggers apoptosis by down-regulating embryonic AKT.

OBJECTIVE: To study the effect of polyethylene glycated leukemia inhibitory factor (LIF) antagonist (PEGLA) in the human blastocyst viability and implantation process. DESIGN: In vitro study. SETTING: University hospital and research laboratory. PATIENT(S): Endometrial biopsy samples from fertile donors (n = 20), and surplus, frozen, good-quality human embryos obtained from an in vitro fertilization (IVF) clinic that survived thawing (n = 51). INTERVENTION(S): Timed human endometrial biopsy on the day of luteinizing hormone peak + 4 days (LH + 4). MAIN OUTCOME MEASURE(S): Human embryo attachment rate, embryo quality, and expression of AKT and caspase-3. RESULT(S): PEGLA significantly reduced the embryo attachment rate to the endometrial construct. It decreased both mRNA and protein for LIF in the endometrial construct. Inhibition of embryonic LIF triggered apoptosis. Analysis of these blastocysts by immunofluorescence and real-time polymerase chain reaction showed a down-regulation in AKT activation and an increase in caspase-3 activation compared with the control group of blastocysts. CONCLUSION(S): The LIF inhibitor PEGLA could be a potential nonsteroidal fertility-regulating agent in humans. It acts on endometrial epithelial cells by down-regulating endometrial epithelial LIF. Inhibition of blastocyst LIF decreased its cell survival factor p-AKT and increased apoptosis (cleaved caspase-3). This highlights that embryonic LIF is vital for human embryo implantation.

Using anti-Müllerian hormone to identify a good prognosis group in women of advanced reproductive age.

BACKGROUND: The probability of pregnancy after in vitro fertilisation (IVF) declines with age in parallel with a reduction in the ovarian reserve. However, there is considerable variation in the ovarian reserve in women of advanced reproductive age; so to give such women accurate advice about the prospects of treatment success, factors other than age must be considered. Anti-Müllerian hormone (AMH) has been shown to be a good indicator of ovarian reserve, and its utility is explored in this paper. AIMS: To determine the utility of AMH serum levels for prediction of ovarian response to gonadotropin stimulation and outcome in IVF in women of advanced reproductive age. METHODS: The material consists of 127 women with a median age of 42 years (range 39-46) having had their first cycle of IVF/intracytoplasmic sperm injection (ICSI) treatment from November 2006 to December 2008. During this period, a total of 772 oocyte retrievals and 715 embryo transfers were performed at the clinic (median age 36.4 years). AMH was analysed with the Beckman Coulter DSL ELISA. Agonist and antagonist protocols were used and monitored by ultrasound and oestradiol; embryo transfer was performed on day 2, 3 or 5 of culture. RESULTS: The lower the AMH, the higher the risk of cycle cancellation, low oocyte yield and treatment failure. Women with a serum AMH above 8.6 pmol/L had a good chance of achieving live birth after IVF/ICSI treatment. CONCLUSIONS: Anti-Müllerian hormone is useful for identifying a good prognosis group in women of advanced reproductive age.

Prediction of embryo developmental potential and pregnancy based on early stage morphological characteristics.

OBJECTIVE: To analyze the association between morphological details at different stages of culture with blastocyst development, with an aim to improve selection for transfer. DESIGN: Retrospective audit of data. SETTING: Tertiary referral center and university hospital. PATIENT(S): Two hundred sixty-eight couples underwent 357 treatment cycles. INTERVENTION(S): Oocyte pickups for IVF or intracytoplasmic sperm injection (ICSI) after ovarian stimulation. Embryos were individually cultured and examined on days 0-2 for morphological details and developmental characteristics, and selected for transfer, freezing, or further culture. MAIN OUTCOME MEASURES: The association of blastocyst development and pregnancy with morphological characteristics. RESULT(S): Five morphological characteristics (appearance of the cytoplasm, pronuclei and nucleoli, cytoplasmic deficit, and developmental rate) showed the strongest association with blastocyst development. By combining information from all days of culture into a cumulative score, prediction was greatly improved, compared to only using day 2 morphology. Cytoplasmic dysmorphisms of the oocyte, including accumulation of smooth endoplasmic reticulum, were associated with poor developmental performance. Differential weighting of these characteristics was included in a new embryo scoring system, which showed a strong correlation with implantation. CONCLUSION(S): Weighting individual morphological characteristics of zygotes and embryos and combining them into a cumulative embryo score can improve selection of embryos for transfer.

Critical evaluation of human blastocysts for assisted reproduction techniques and embryonic stem cell biotechnology.

Critical examination of 30 blastocysts by transmission electron microscopy (TEM) reveals cellular features not usually evident, including abnormalities of cell structure and aberrations such as multinucleation, internal fragmentation, phagocytic or degenerating cells. Invariably, such blastocysts are inactive and delay or fail to expand and hatch in vitro. Hatching seems to be a major problem in ageing blastocysts due to inactivity of the surface epithelium of trophoblast cells that do not stretch and expand. These lack surface microvilli and contractile tonofilaments that anchor on to specialized cell junctions such as desmosomes. Trophoblast expansion and consequent thinning of the zona is a prerequisite to proper hatching aided by the hydrostatic pressure in the blastocoele and by specialized cells at hatching points. Proper assessment of the inner cell mass is required if a healthy population of cells is to be harvested for embryonic stem cell culture. An inactive blastocyst is obviously not good material and could have a defective inner cell mass (ICM). Normally approximately 3-5% of cells are mitotic in blastocysts and arrested cell division is also an indicator of inactivity. An attempt has been made to evaluate blastocyst internal structure for both assisted reproduction techniques and embryonic stem cell biotechnology.

Mechanics of human blastocyst hatching in vitro.

Critical examination of 30 blastocysts by transmission electron microscopy at various stages of blastulation and hatching, has revealed the presence of specialized, plump, trophoblastic cells at the points of hatching, which seem to aid in initial breaking of the zona pellucida (ZP) and then widen its opening to permit the progressive emergence of the embryo in amoeboid fashion, when it acquires a characteristic dumb-bell shape. These cells are named 'zona-breaker' cells and their characteristics are described. Normally, trophoblast cells in expanding blastocysts are flattened (squamous), forming a continuous robust epithelium with specialized cell junctions. Bundles of tonofilaments anchor onto desmosomes, forming a terminal web. Proper expansion of blastocysts by intake of fluid into the blastocoele causes an increase in internal hydrostatic pressure that stretches the trophoblast epithelium leading to an enlargement of its volume two- to three-fold, consequently thinning the zona prior to hatching. This is an important prerequisite to normal hatching. The blastocysts usually hatch out at the pole opposite the inner cell mass (ICM), though a few hatch out at the embryonal pole or elsewhere. In all cases zona-breakers seem to play a vital role in the hatching process.