Impact of letrozole co-treatment during ovarian stimulation on oocyte yield, embryo development, and live birth rate in women with normal ovarian reserve: secondary outcomes from the RIOT trial.

STUDY QUESTION: Does letrozole (LZ) co-treatment during ovarian stimulation with gonadotropins for in IVF impact follicle recruitment, oocyte number and quality, embryo quality, or live birth rate (LBR)? SUMMARY ANSWER: No impact of LZ was found in follicle recruitment, number of oocytes, quality of embryos, or LBR. WHAT IS KNOWN ALREADY: Multi-follicle stimulation for IVF produces supra-physiological oestradiol levels. LZ is an aromatase inhibitor that lowers serum oestradiol thus reducing negative feedback and increasing the endogenous gonadotropins in both the follicular and the luteal phases, effectively normalizing the endocrine milieu during IVF treatment. STUDY DESIGN, SIZE, DURATION: Secondary outcomes from a randomized, double-blind placebo-controlled trial (RCT) investigating once-daily 5 mg LZ or placebo during stimulation for IVF with FSH. The RCT was conducted at four fertility clinics at University Hospitals in Denmark from August 2016 to November 2018 and pregnancy outcomes of frozen-thawed embryo transfers (FET) registered until May 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: One hundred fifty-nine women with expected normal ovarian reserve (anti-Müllerian hormone 8-32 nmol/l) were randomized to either co-treatment with LZ (n = 80) or placebo (n = 79). In total 1268 oocytes were aspirated developing into 386 embryos, and morphology and morphokinetics were assessed. One hundred twenty-nine embryos were transferred in the fresh cycle and 158 embryos in a subsequent FET cycle. The effect of LZ on cumulative clinical pregnancy rate (CPR), LBR, endometrial thickness in the fresh cycle, and total FSH consumption was reported. MAIN RESULTS AND THE ROLE OF CHANCE: The proportion of usable embryos of retrieved oocytes was similar in the LZ group and the placebo group with 0.31 vs 0.36 (mean difference (MD) -0.05, 95% CI (-0.12; 0.03), P = 0.65). The size and number of aspirated follicles at oocyte retrieval were similar with 11.8 vs 10.3 follicles per patient (MD 1.5, 95% CI (-0.5; 3.1), P = 0.50), as well as the number of retrieved oocytes with 8.0 vs 7.9 oocytes (MD 0.1, 95% CI (-1.4; 1.6), P = 0.39) in the LZ and placebo groups, respectively. The chance of retrieving an oocyte from the 13 to 16 mm follicles at trigger day was 66% higher (95% CI (24%; 108%), P = 0.002) in the placebo group than in the LZ group, whilst the chance of retrieving an oocyte from the ≥17 mm follicles at trigger day was 50% higher (95% CI (2%; 98%), P = 0.04) in the LZ group than in the placebo group. The proportion of fertilized oocytes with two-pronuclei per retrieved oocytes or per metaphase II oocytes (MII) (the 2PN rates) were similar regardless of fertilization with IVF or ICSI with 0.48 vs 0.57 (MD -0.09, 95% CI (-0.24; 0.04), P = 0.51), and 0.62 vs 0.64 (MD -0.02, 95% CI (-0.13; 0.07), P = 0.78) in the LZ and placebo groups, respectively. However, the MII rate in the ICSI group was significantly lower with 0.75 vs 0.88 in the LZ vs the placebo group (MD -0.14, 95% CI (-0.22; -0.06), P = 0.03). Blastocysts on Day 5 per patient were similar with 1.5 vs 2.0, P = 0.52, as well as vitrified blastocysts per patient Day 5 with 0.8 vs 1.2 in (MD -0.4, 95% CI (-1.0; 0.2), P = 0.52) and vitrified blastocysts per patient Day 6 with 0.6 vs 0.6 (MD 0, 95% CI (-0.3; 0.3), P = 1.00) in the LZ vs placebo group, respectively. Morphologic evaluation of all usable embryos showed a similar distribution in 'Good', 'Fair', and 'Poor', in the LZ vs placebo group, with an odds ratio (OR) of 0.8 95% CI (0.5; 1.3), P = 0.68 of developing a better class embryo. Two hundred and ninety-five of the 386 embryos were cultured in an embryoscope. Morphokinetic annotations showed that the odds of having a high KIDscore™ D3 Day 3 were 1.2 times higher (CI (0.8; 1.9), P = 0.68) in the LZ group vs the placebo group. The CPR per transfer was comparable with 31% vs 39% (risk-difference of 8%, 95% CI (-25%; 11%), P = 0.65) in the LZ and placebo group, respectively, as well as CPR per transfer adjusted for day of transfer, oestradiol and progesterone levels at trigger, progesterone levels mid-luteal, and number of oocytes retrieved (adjusted OR) of 0.8 (95% CI (0.4; 1.6), P = 0.72). Comparable LBR were found per transfer 28% vs 37% (MD -9%, 95% CI (-26%; 9%), P = 0.60) and per randomized women 24% vs 30% (MD of -6%, CI (-22%; 8%), P = 0.60) in the LZ group and placebo group, respectively. Furthermore, 4.8 years since the last oocyte aspiration, a total of 287 of 386 embryos have been transferred in the fresh or a subsequently FET cycle, disclosing the cumulative CPR, which is similar with 38% vs 34% (MD 95% CI (8%; 16%), P = 0.70) in the LZ vs placebo group. LIMITATIONS, REASONS FOR CAUTION: Both cleavage stage and blastocyst transfer and vitrification were permitted in the protocol, making it necessary to categorize their quality and pool the results. The study was powered to detect hormonal variation but not embryo or pregnancy outcomes. WIDER IMPLICATIONS OF THE FINDINGS: The similar utilization rate and quality of the embryos support the use of LZ co-treatment for IVF with specific indication as fertility preservation, patients with previous cancer, or poor responders. The effect of LZ on mature oocytes from different follicle sizes and LBRs should be evaluated in a meta-analysis or a larger RCT. STUDY FUNDING/COMPETING INTEREST(S): Funding was received from EU Interreg for ReproUnion, Sjaelland University Hospital, Denmark, Ferring Pharmaceuticals, and Gedeon Ricther. Roche Diagnostics contributed with assays. A.P. has received grants from Ferring, Merck Serono, and Gedeon Richter, consulting fees from Preglem, Novo Nordisk, Ferring, Gedeon Richter, Cryos, & Merck A/S, speakers fees from Gedeon Richter, Ferring, Merck A/S, Theramex, & Organon, and travel support from Gedeon Richter. The remaining authors declare that they have no competing interests in the research or publication. TRIAL REGISTRATION NUMBERS: NCT02939898 and NCT02946684.

Conjoined twins after single blastocyst transfer: a case report including detailed time-lapse recording of the earliest embryogenesis, from zygote to expanded blastocyst.

Conjoined twins are estimated to occur in 1:50 000 pregnancies. Eighteen cases of pregnancies achieved by ART have been published of which three were achieved after single embryo transfer, allowing discussion of embryo characteristics. We report, to the best of our knowledge, the first case of parapagus conjoined twins after ART. Furthermore, this is the first report of conjoined twins with detailed morphokinetics of the earliest embryogenesis from zygote to expanded and hatched blastocyst stage. The case zygote had three refractile bodies, which were all allocated to one blastomere at first cleavage following an asynchronous pronuclei fading. Within 2 h, this blastomere cleaved to four and fragmented. The remaining blastomere cleaved symmetrically and regularly and a blastocyst (score: 4AB) was vitrified 120 h after IVF. Pregnancy was achieved following a frozen-thawed single blastocyst transfer. The etiopathogenetic mechanism of the origin of conjoined twins is unknown and several hypotheses exist. The morphokinetics in the present case and morphology of other reported cases will be discussed in this context.

Impact of letrozole co-treatment during ovarian stimulation with gonadotrophins for IVF: a multicentre, randomized, double-blinded placebo-controlled trial.

STUDY QUESTION: Does letrozole co-treatment during ovarian stimulation with gonadotrophins for IVF reduce the proportion of women with premature progesterone levels above 1.5 ng/ml at the time of triggering final oocyte maturation? SUMMARY ANSWER: The proportion of women with premature progesterone above 1.5 ng/ml was not significantly affected by letrozole co-treatment. WHAT IS KNOWN ALREADY: IVF creates multiple follicles with supraphysiological levels of sex steroids interrupting the endocrine milieu and affects the window of implantation. Letrozole is an effective aromatase inhibitor, normalizing serum oestradiol, thereby ameliorating some of the detrimental effects of IVF treatment. STUDY DESIGN, SIZE, DURATION: A randomized, double-blinded placebo-controlled trial investigated letrozole intervention during stimulation for IVF with FSH. The trial was conducted at four fertility clinics at University Hospitals in Denmark from August 2016 to November 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: A cohort of 129 women with expected normal ovarian reserve (anti-Müllerian hormone 8-32 nmol/l) completed an IVF cycle with fresh embryo transfer and received co-treatment with either 5 mg/day letrozole (n = 67) or placebo (n = 62), along with the FSH. Progesterone, oestradiol, FSH, LH and androgens were analysed in repeated serum samples collected from the start of the stimulation to the mid-luteal phase. In addition, the effect of letrozole on reproductive outcomes, total FSH consumption and adverse events were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: The proportion of women with premature progesterone >1.5 ng/ml was similar (6% vs 0% (OR 0.0, 95% CI [0.0; 1.6], P = 0.12) in the letrozole versus placebo groups, respectively), whereas the proportion of women with mid-luteal progesterone >30 ng/ml was significantly increased in the letrozole group: (59% vs 31% (OR 3.3, 95% CI [1.4; 7.1], P = 0.005)). Letrozole versus placebo decreased oestradiol levels on the ovulation trigger day by 68% (95% CI [60%; 75%], P < 0.0001). Other hormonal profiles, measured as AUC, showed the following results. The increase in LH in the letrozole group versus placebo group was 38% (95% CI [21%; 58%], P < 0.0001) and 34% (95% CI [11%; 61%], P = 0.006) in the follicular and luteal phases, respectively. In the letrozole group versus placebo group, testosterone increased by 79% (95% CI [55%; 105%], P < 0.0001) and 49% (95% CI [30%; 72%], P < 0.0001) in the follicular and luteal phases, respectively. In the letrozole group versus placebo group, the increase in androstenedione was by 85% (95% CI [59%; 114%], P < 0.0001) and 69% (95% CI [48%; 94%], P < 0.0001) in the follicular and luteal phases, respectively. The ongoing pregnancy rate was similar between the letrozole and placebo groups (31% vs 39% (risk-difference of 8%, 95% CI [-25%; 11%], P = 0.55)). No serious adverse reactions were recorded in either group. The total duration of exogenous FSH stimulation was 1 day shorter in the intervention group, significantly reducing total FSH consumption (mean difference -100 IU, 95% CI [-192; -21], P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Late follicular progesterone samples were collected on the day before and day of ovulation triggering for patient logistic considerations, and the recently emerged knowledge about diurnal variation of progesterone was not taken into account. The study was powered to detect hormonal variations but not differences in pregnancy outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Although the use of letrozole has no effect on the primary outcome, the number of women with a premature increase in progesterone on the day of ovulation triggering, the increased progesterone in the mid-luteal phase due to letrozole may contribute to optimizing the luteal phase endocrinology. The effect of letrozole on increasing androgens and reducing FSH consumption may be used in poor responders. However, the effect of letrozole on implantation and ongoing pregnancy rates should be evaluated in a meta-analysis or larger randomized controlled trial (RCT). STUDY FUNDING/COMPETING INTEREST(S): Funding was received from EU Interreg for ReproUnion and Ferring Pharmaceuticals, and Roche Diagnostics contributed with assays. N.S.M. and A.P. have received grants from Ferring, Merck Serono, Anecova and Gedeon Richter, and/or personal fees from IBSA, Vivoplex, ArtPred and SPD, outside the submitted work. The remaining authors have no competing interests. TRIAL REGISTRATION NUMBERS: NCT02939898 and NCT02946684. TRIAL REGISTRATION DATE: 15 August 2016. DATE OF FIRST PATIENT'S ENROLMENT: 22 August 2016.

Maternal exome analysis for the diagnosis of oocyte maturation defects and early embryonic developmental arrest.

RESEARCH QUESTION: Can a methodology be developed for case selection and whole-exome sequencing (WES) analysis of women who are infertile owing to recurrent oocyte maturation defects (OOMD) and/or preimplantation embryo lethality (PREMBL)? DESIGN: Data were collected from IVF patients attending the Istanbul Memorial Hospital (2015-2021). A statistical methodology to identify infertile endophenotypes (recurrent low oocyte maturation rate, low fertilization rate and preimplantation developmental arrest) was developed using a large IVF dataset (11,221 couples). Twenty-eight infertile women with OOMD/PREMBL were subsequently enrolled for WES on their genomic DNA. Pathogenic variants were prioritized using a custom-made bioinformatic pipeline set to minimize false-positive discoveries through resampling in control cohorts (the Human Genome Diversity Project and 1343 whole-exome sequences from oocyte donors). Individual single-cell RNA sequencing data from 18 human metaphase II (MII) oocytes and antral granulosa cells was used for genome-wide validation. WES and bioinformatics were performed at Igenomix and the National Research Council, Italy. RESULTS: Variant prioritization analysis identified 265 unique variants in 248 genes (average 22.4 per sample). Of the genes harbouring high-impact variants 78% were expressed by MII oocytes and/or antral granulosa cells, significantly higher than for random sample of controls (odds ratio = 5, Fisher's exact P = 0.0004). Seven of the 28 women (25%) were homozygous carriers of missense pathogenic variants in known candidate genes for OOMD/PREMBL, including PATL2, NLRP5 (n = 2),TLE6, PADI6, TUBB8 and TRIP13. Furthermore, novel gene-disease associations were identified. In fact, one woman with a low oocyte maturation rate was a homozygous carrier of high-impact variants in ENSA, an essential gene for prophase I meiotic transition in mice. CONCLUSIONS: This analytical framework could reveal known and new genes associated with isolated recurrent OOMD/PREMBL, providing essential indications for scaling this strategy to larger studies.

Preimplantation genetic testing legislation and accessibility in the Nordic countries.

INTRODUCTION: Assisted reproduction technologies are being rapidly developed and implementation of preimplantation genetic testing (PGT) has allowed patients with genetic disorders to initiate pregnancies while minimizing or eliminating the risk of transmitting these disorders to their offspring. Testing for numeric chromosomal anomalies has been proposed as a way to increase efficacy in assisted reproduction; however, this remains disputed. Legislation is lagging behind the rapid developments in this field. MATERIAL AND METHODS: We conducted a structured online survey of legislation and accessibility to preimplantation genetic testing in the Nordic countries to compare the regulation and uptake of this technique. The survey was designed and answered by the authors. RESULTS: Key elements in the regulation of preimplantation testing for monogenic disorders and structural rearrangements are similar in the Nordic countries, although accessibility varies since only Denmark, Finland, and Sweden have national clinics offering treatment. In addition, Denmark and Finland have private clinics offering PGT. Regulation is the most stringent in Norway where a national board evaluates all couples seeking treatment. Treatment volumes vary between the Nordic countries, with Norway and Finland having lowest treatment numbers. Preimplantation genetic testing for aneuploidy in the embryo varies between the Nordic countries: Finland and Iceland allow this form of treatment, Denmark and Sweden offer it only in the form of a research protocol, and Norway does not allow it at all. Therefore the number of treatment cycles involving testing for embryo aneuploidy are lower in the Nordic countries than in other countries where this treatment option is more common. CONCLUSIONS: Science needs to inform politics regarding the rapidly evolving field of reproductive medicine and we recommend harmonization of legislation and accessibility between the Nordic countries.

Preimplantation genetic testing practices in the Nordic countries.

INTRODUCTION: Preimplantation genetic testing (PGT) is growing in importance and volume internationally. International societies such as the European Society for Human Reproduction and Embryology compile international results and these data are published in scientific journals. We present the first compilation of practices, quality measuress and outcome data from Nordic clinics performing PGT. MATERIAL AND METHODS: We conducted a structured online survey of PGT practices in the Nordic countries to compare clinical and laboratory techniques, outcomes and quality measures applied in Nordic clinics. The survey was designed by the authors and answered by the authors and members of the study group. The outcome data represents results from 2018. Results and details were clarified through iteration with responding clinics while maintaining anonymity. Response rate in the study was 80%, with 8 of 10 clinics performing PGT responding. RESULTS: Most of the PGT cycles in the Nordic countries are funded through the public healthcare system with University Hospitals performing the majority of treatments, 716/848, or 84.4%, of oocyte retrievals in this dataset. The genetic analyses are in five cases performed by the affiliated local genetic laboratory, and the remaining three consult with large international private enterprise laboratories. Genetic counseling is widely used. Results in the Nordic clinics compare well with international data. Systematic quality control procedures are in place and the larger clinics and laboratories utilize ISO certification or accreditation in the quality management. Automatic witnessing with detailed electronic documentation of laboratory processes is not utilized in the responding clinics, although a majority uses manual witnessing procedures in the laboratory. The outcome after PGT in terms of clinical pregnancy per transfer is around 40% per embryo transfer and compares well with international data. CONCLUSIONS: Preimplantation genetic testing is organized in rather few clinics in the Nordic countries and most of them use local laboratories for genetic analyses of the biopsies. Laboratory procedures are largely in accordance with international guidelines and the outcome after PGT in terms of clinical pregnancy per transfer is comparable to results in international reports.

Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.

PURPOSE: The aim of the present study was to improve the in vitro maturation (IVM) procedure using oocytes from surplus ovarian tissue after fertility preservation. METHODS: Twenty-five patients aged 17-37 years were included in the study. Maturation was compared between oocytes collected in HEPES-buffered medium or saline, and we determined whether transport on ice prior to oocyte collection affected maturation. Two different IVM media were used that were supplemented with and without recombinant human midkine. Mature oocytes were assessed for aneuploidy using next-generation sequencing (NGS). RESULTS: On average, 36 immature oocytes were collected from each patient (range 7-90, N = 895). Oocytes recovered from HEPES-buffered medium matured at a higher rate than oocytes recovered from saline (36% vs 26%, p < 0.01). Ovarian transportation on ice prior to the procedure negatively affected maturation compared with non-transported samples (42% vs 27%, p < 0.01). The addition of midkine improved maturation rate (34% vs 27%, p < 0.05). On average, 11 MII oocytes were obtained per patient (range 1-30). NGS of 53 MII oocytes and their first polar bodies indicated that 64% were euploid. CONCLUSIONS: The study demonstrated unexpectedly high number of immature oocytes collected from surplus ovarian tissue without any stimulation. The overall MII rate was one in three, resulting in a total number of MII oocytes that was similar to the number obtained after ovarian stimulation. If these MII oocytes prove suitable for IVF, they will provide a substantial improvement in fertility preservation for patients and advance IVM as an interesting platform for further improvements in assisted reproduction.

Non-invasive prenatal paternity testing using a standard forensic genetic massively parallel sequencing assay for amplification of human identification SNPs.

Prenatal paternity testing often relies on invasive procedures that cause risk to both the mother and the foetus. Non-invasive, prenatal paternity testing by investigating paternally inherited single nucleotide polymorphisms (SNPs) in cell-free foetal DNA (cffDNA) in maternal plasma was performed at consecutive time points during early gestation. Plasma from 15 pregnant women was investigated at consecutive time points from gestational weeks (GWs) 4-20. The Precision ID Identity Panel and an Ion S5 Sequencer was used to analyse the cffDNA. Paternally inherited foetal SNP alleles were detected from GW7. The median foetal fractions were 0%, 3.9%, 5.1%, 5.2%, and 4.7% at GWs 4, 7, 12, 16, and 20, respectively. The corresponding median numbers of detected paternally inherited foetal autosomal SNP alleles were 0, 3, 9, 10, and 12, respectively. The typical (i.e. geometric mean) paternity indices at GW12 and GW20 were 24 (range 0.0035-8389) and 199 (range 5.1-30,137), respectively. The method is very promising. However, the method can be improved by shortening the lengths of the PCR amplicons and increasing the number of SNPs. To our knowledge, this is the first study to successfully identify paternally inherited foetal SNP alleles at consecutive time points in early gestation independently of the foetal gender.

Dynamics of IGF signalling during the ovulatory peak in women undergoing ovarian stimulation.

CONTEXT: IGF signalling is known to affect human ovarian follicular function during growth and development. However, the role of the IGF system is unknown during the ovulatory peak, which is characterized by profound changes in granulosa cell (GCs) mitosis and function. OBJECTIVE: How is the IGF system expressed and regulated during the midcycle surge in women? DESIGN: Follicular fluid (FF) and granulosa cells (GCs) were collected during the ovulatory peak from two specific time-points. One sample was obtained before oocyte pick up (OPU): before ovulation trigger (OT) (T = 0 h) or at 12, 17, or 32 h after OT, and one sample was obtained at OPU 36 h after OT. SETTING: University hospital. PATIENTS/PARTICIPANTS: Fifty women undergoing ovarian stimulation were included. MAIN OUTCOME MEASURE: Gene expression profiles were assessed by microarray analysis of GCs. IGF-related proteins in the FF were assessed by using immunoassays or by determination of activity with a proteinase assay. RESULTS: Expression of proteins promoting IGF activity (i.e., IGF2, PAPPA, and IRS1) together with proliferation markers were downregulated on a transcriptional level in GCs after OT, whereas proteins inhibiting the IGF signal (i.e., IGFBPs, IGF2R, and STC1) were upregulated. STC1 gene expression and protein levels were greatly upregulated after OT with a parallel steep downregulation of PAPP-A proteolytic activity. CONCLUSIONS: These data suggest that downregulation of IGF signalling mediated by increased STC1 expression is instrumental for the sudden cessation in GC proliferation and onset of differentiation during the ovulatory peak.

The dormant and the fully competent oocyte: comparing the transcriptome of human oocytes from primordial follicles and in metaphase II.

Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P < 0.001) representing functional categories such as cell cycle regulation, DNA protection and epigenetics, with representative genes validated by qPCR analysis. Dominating canonical pathways in the oocytes from primordial follicles were androgen, estrogen receptor, glucocorticoid receptor and PI3K/AKT signaling (P < 0.001). In the MII, mitotic roles of polo-like kinases, estrogen receptor, JAK/Stat signaling (P < 0.001) and the ERK/MAPK (P < 0.01) signaling were enriched. Some of the highly differentially expressed genes were completely new in human reproduction (CDR1, TLC1A, UHRF2) while other genes [ABO, FOLR1 (folate receptor), CHRNA3 (nicotine receptor)] may relate to clinical observations as diverse as premature ovarian failure, folic acid deficiency and smoking affecting female fertility. The in silico analysis identified novel reproduction-associated genes and highlighted molecular mechanisms and pathways associated with the unique functions of the human oocyte in its two extremes during folliculogenesis. The data provides a fundamental basis for future functional studies in regulation of human oogenesis.

A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator.

PURPOSE: Time-lapse monitoring allows for a flexible embryo evaluation and potentially provides new dynamic markers of embryo competence. Before introducing time-lapse monitoring in a clinical setting, the safety of the instrument must be properly documented. Accordingly, the aim of this study was to evaluate the safety of a commercially available time-lapse incubator. METHODS: In a two center, randomized, controlled, clinical trial 676 oocytes from 59 patients in their 2nd or third treatment cycle, age <38 years and ≥ 8 oocytes retrieved were cultured in the time-lapse incubator or in a conventional incubator. The primary outcome was proportion of 4-cell embryos on day 2. Secondary outcomes were proportion of 7-8 cell embryos on day 3 and proportion of blastocysts on day 5. Implantation pregnancy rates were registered based on presence of fetal heart activity visualized by ultrasound 8 weeks after embryo transfer. RESULTS: No significant difference was found between the time-lapse incubator (TLI) and conventional incubator (COI) in proportion of 4-cell embryos on day 2 irrespective of whether data was analyzed according to ITT (RR(TLI/COI): 0.81 (0.65; 1.02)) or PP (RR(TLI/COI): 0.80 (0.63; 1.01)). Nor were any significant differences detected in the secondary endpoints; i.e. proportion of 7-8-cell embryos on day three ITT (RR(TLI/COI): 0.96 (0.73; 1.26)); PP (RR(TLI/COI): 0.95 (0.72; 1.26)) and proportion of blastocysts on day five ITT (RR(TLI/COI): 1.09 (0.84; 1.41)); PP (RR(TLI/COI): 1.09 (0.83: 1.41)). We found no differences in clinical pregnancy rate or implantation rate. CONCLUSION: Culture in the time-lapse incubator supports embryonic development equally to a conventional incubator.

Human Oocyte Morphology and Outcomes of Infertility Treatment: a Systematic Review.

Oocyte morphology assessment is easy to implement in any laboratory with possible quality grading prior to fertilization. At present, comprehensive oocyte morphology scoring is not performed as a routine procedure. However, it may augment chances for successful treatment outcomes if a correlation with certain dysmorphisms can be proven. In order to determine a correlation between oocyte morphology and treatment outcome, we performed a systematic search in PubMed and Cochrane Controlled Trials Register following PRISMA guidelines. A total of 52 articles out of 6,755 search results met the inclusion criteria. Dark colour of the cytoplasm (observed with an incidence rate of 7%), homogeneous granularity of the cytoplasm (19%) and ovoid shape of oocytes (7%) appeared to have no influence on treatment outcome. Abnormalities such as refractile bodies (10%), fragmented first polar body (37%), dark zona pellucida (9%), enlarged perivitelline space (18%) and debris in it (21%) are likely to affect the treatment outcome to some extent. Finally, cytoplasmic vacuoles (4%), centrally located cytoplasmic granularity (12%) and clusters of smooth endoplasmic reticulum (4%) negatively impact infertility treatment outcomes. Nonetheless, morphological assessment is informative rather than predictive. Adding oocyte morphology to the artificial intelligence (AI)-driven selection process may improve the precision of the algorithms. Oocyte morphology assessment can be especially useful in oocyte donation cycles, during oocyte freezing for fertility preservation and finally, objective oocyte scoring can be important in cases of very poor treatment outcome as a tool for explanation of results to the patient.

Microdissection Testicular Sperm Extraction Versus Multiple Needle-pass Percutaneous Testicular Sperm Aspiration in Men with Nonobstructive Azoospermia: A Randomized Clinical Trial.

BACKGROUND: Surgical extraction of testicular spermatozoa is needed in men with nonobstructive azoospermia (NOA) who wish to become biological fathers. Based on available uncontrolled studies with unspecific patient selection, microdissection testicular sperm extraction (mTESE), having a sperm retrieval rate (SRR) of 50%, is considered the most efficient sperm retrieval procedure. However, no randomized clinical trials for comparison of different sperm retrieval procedures exist. Testicular sperm aspiration (TESA) is simple and commonly used, and we hypothesized that this technique using multiple needle passes would give similar SRRs to mTESE. OBJECTIVE: To compare mTESE and multiple needle-pass TESA in men with NOA. DESIGN, SETTING, AND PARTICIPANTS: A randomized clinical trial was performed between June 2017 and April 2021, with inclusion of 100 men with NOA from four centers in Denmark and Sweden. All participants received treatment at the same institution. INTERVENTION: Participants were randomized to mTESE (n = 49) or multiple needle-pass TESA (n = 51). Patients with failed multiple needle-pass TESA proceeded directly to salvage mTESE. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was SRR. Secondary outcomes included complications and changes in reproductive hormones after surgery. RESULTS AND LIMITATIONS: Spermatozoa were retrieved in 21/49 (43%) men after mTESE and in 11/51 (22%) men after multiple needle-pass TESA (rate difference -0.21; 95% confidence interval -0.39 to -0.03; p = 0.02). The combined SRR for multiple needle-pass TESA + salvage mTESE was 15/51 (29%). No complications occurred after multiple needle-pass TESA only, while 5/89 (6%) men having mTESE experienced a complication requiring surgical intervention. Overall, no statistically significant differences in reproductive hormones were observed between groups after 6 mo. Limitations include the low number of patients in secondary outcome data. CONCLUSIONS: In direct comparison, SRR was higher in mTESE than in multiple needle-pass TESA. PATIENT SUMMARY: Men with azoospermia need surgical extraction of spermatozoa to become biological fathers. In this randomized trial, we compared two surgeries (microdissection testicular sperm extraction [mTESE] and testicular sperm aspiration [TESA]) and found that mTESE gives a higher sperm retrieval rate than multiple needle-pass TESA.

Is paternal age associated with transfer day, developmental stage, morphology, and initial hCG-rise of the competent blastocyst leading to live birth? A multicenter cohort study.

In this study we investigated whether age of men undergoing assisted reproductive technology (ART) treatment was associated with day of transfer, stage, morphology, and initial hCG-rise of the competent blastocyst leading to a live birth? The design was a multicenter historical cohort study based on exposure (age) and outcome data (blastocyst stage and morphology and initial hCG-rise) from men whose partner underwent single blastocyst transfer resulting in singleton pregnancy/birth. The ART treatments were carried out at sixteen private and university-based public fertility clinics. We included 7246 men and women, who between 2014 and 2018 underwent controlled ovarian stimulation (COS) or Frozen-thawed Embryo Transfer (FET) with a single blastocyst transfer resulting in singleton pregnancy were identified. 4842 men with a partner giving birth were included, by linking data to the Danish Medical Birth Registry. We showed that the adjusted association between paternal age and transfer day in COS treatments was OR 1.06, 95% CI (1.00;1.13). Meaning that for every increase of one year, men had a 6% increased probability that the competent blastocyst was transferred on day 6 compared to day 5. Further we showed that the mean difference in hCG values when comparing paternal age group 30-34, 35-39 and 40-45 with the age group 25-29 in those receiving COS treatment, all showed significantly lower adjusted values for older men. In conclusion we hypothesize that the later transfer (day 6) in female partners of older men may be due to longer time spent by the oocyte to repair fragmented DNA of the sperm cells, which should be a focus of future research in men.

In vitro fertilisation (IVF) versus intracytoplasmic sperm injection (ICSI) in patients without severe male factor infertility: study protocol for the randomised, controlled, multicentre trial INVICSI.

INTRODUCTION: Over the last decades, the use of intracytoplasmic sperm injection (ICSI) has increased, even among patients without male factor infertility. The increase has happened even though there is no evidence to support that ICSI results in higher live birth rates compared with conventional in vitro fertilisation (IVF) in cases with nonmale factor infertility. The lack of robust evidence on an advantage of using ICSI over conventional IVF in these patients is problematic since ICSI is more invasive, complex and requires additional resources, time and effort. Therefore, the primary objective of the IVF versus ICSI (INVICSI) study is to determine whether ICSI is superior to standard IVF in patients without severe male factor infertility. The primary outcome measure is first live birth from fresh and frozen-thawed transfers after one stimulated cycle. Secondary outcomes include fertilisation rate, ongoing pregnancy rate, birth weight and congenital anomalies. METHODS AND ANALYSIS: This is a two-armed, multicentre, randomised, controlled trial. In total, 824 couples/women with infertility without severe male factor will be recruited and allocated randomly into two groups (IVF or ICSI) in a 1:1 ratio. Participants will be randomised in variable block sizes and stratified by trial site and age. The main inclusion criteria are (1) no prior IVF/ICSI treatment, (2) male partner sperm with an expected count of minimum 2 million progressive motile spermatozoa following density gradient purification on the day of oocyte pick up and (3) age of the woman between 18 and 42 years. ETHICS AND DISSEMINATION: The study will be performed in accordance with the ethical principles in the Helsinki Declaration. The study is approved by the Scientific Ethical Committee of the Capital Region of Denmark. Study findings will be presented, irrespectively of results at international conferences and submitted for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04128904. Pre-results.

[Preimplantation genetic testing].

Preimplantation genetic testing (PGT) for known familial monogenetic disease (PGT-M) or structural chromosomal rearrangements (PGT-SR) has evolved into a well-established alternative to prenatal diagnosis. PGT significantly reduces the risk of a pregnancy with an affected foetus. Screening for aneuploidy (PGT-A) used as an add-on to standard IVF treatment of infertile couples is widely used internationally, although its benefit is highly debated. PGT combines genetic counselling and testing with assisted reproductive technology including ovarian stimulation, egg retrieval, and embryo biopsy, as discussed in this review.

Identification of a unique epigenetic profile in women with diminished ovarian reserve.

OBJECTIVE: To investigate whether epigenetic profiles of mural granulosa cells (MGC) and leukocytes from women with diminished ovarian reserve (DOR) differ from those of women with normal or high ovarian reserve. DESIGN: Prospectively collected material from a multicenter cohort of women undergoing fertility treatment. SETTING: Private and university-based facilities for clinical services and research. PATIENT(S): One hundred and nineteen women of various ages and ovarian reserve status (antimüllerian hormone level) who provided blood samples and MGC. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Measures of epigenetic aging rates from whole-genome methylation array data: DNA methylation variability, age acceleration, DNA methylation telomere length estimator (DNAmTL), and accumulation of epimutations. RESULT(S): Comparison of DOR or high ovarian reserve samples to controls (normal ovarian reserve) showed differential methylation variability between DOR and normal samples at 4,199 CpGs in MGC, and 447 between high and normal (false-discovery rate < 0.05). Variable sites in MGC from DOR were enriched in regions marked with the repressive histone modification H3K27me3, and also included genes involved in folliculogenesis, such as insulin growth factor 2 (IGF2) and antimüllerian hormone (AMH). Regardless of ovarian reserve, very few signals were detected in leukocytes, and no overlaps with those in MGC were found. Furthermore, we found a higher number of epimutations in MGC from women with DOR (Kruskal-Wallis test, difference in mean = 3,485). CONCLUSION(S): The somatic cells of human ovarian follicles have a distinctive epigenetic profile in women with DOR. A high frequency of epimutations suggests premature aging. Ovarian reserve status was not reflected in the leukocyte epigenetic profile.

Embryo Morphokinetics and Blastocyst Development After GnRH Agonist versus hCG Triggering in Normo-ovulatory Women: a Secondary Analysis of a Multicenter Randomized Controlled Trial.

Gonadotropin-releasing hormone agonist (GnRHa) for final oocyte maturation, along with vitrification of all usable embryos followed by transfer in a subsequent frozen-thawed cycle, is the most effective strategy to avoid ovarian hyperstimulation syndrome (OHSS). However, less is known about the ovulation induction triggers effect on early embryo development and blastocyst formation. This study is a secondary analysis of a multicenter, randomized controlled trial, with the aim to compare embryo development in normo-ovulatory women, randomized to GnRHa or human chorionic gonadotropin (hCG) trigger. In all, 4056 retrieved oocytes were observed, 1998 from the GnRHa group (216 women) and 2058 from the hCG group (218 women). A number of retrieved oocytes, mature and fertilized oocytes, and high-quality embryos and blastocysts were similar between the groups. A sub-analysis in 250 women enrolled at the main trial site including 2073 oocytes was conducted to compare embryo morphokinetics and cleavage patterns with EmbryoScope time-lapse system. In total, 1013 oocytes were retrieved from the GnRHa group (124 women) and 1060 oocytes were retrieved from the hCG group (126 women). Morphokinetic parameters and cleavage patterns were comparable between the groups. However, embryos derived from the GnRHa group were less likely to perform rolling during their development than the embryos from the hCG trigger group (OR = 0.41 (95%CI 0.25; 0.67), p-value 0.0003). The comparable results on embryo development and utilization rates between the GnRHa and hCG triggers is of clinical relevance to professionals and infertile patients, when GnRHa trigger and freeze-all is performed to avoid OHSS development. ClinicalTrials.gov Identifier: NCT02746562.

Association between women's age and stage, morphology, and implantation of the competent blastocyst: a multicenter cohort study.

OBJECTIVE: To study if the age of women undergoing assisted reproductive technology treatment associates with stage, morphology, and implantation of the competent blastocyst. DESIGN: Multicenter historical cohort study based on exposure (age) and outcome data (blastocyst stage and morphology and initial human chorionic gonadotrophin [hCG] rise) from women undergoing single blastocyst transfer resulting in singleton pregnancy/birth. SETTING: Sixteen private and university-based facilities. PATIENT(S): In this study, 7,246 women who, between 2014 and 2018, underwent controlled ovarian stimulation (COS) or frozen-thawed embryo transfer (FET) with a single blastocyst transfer resulting in singleton pregnancy were identified. Linking data to the Danish Medical Birth Registry resulted in a total of 4,842 women with a live birth being included. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The competent blastocyst development stage (1-6), inner cell mass (A, B, C), trophectoderm (A, B, C), and initial serum hCG value. RESULT(S): Adjusted analysis of age and stage in COS treatments showed that for every 1-year increase in age there was a 5% reduced probability of the competent blastocyst assessed as being in a high stage at transfer. Comparison between hCG values in women 18-24 years and 25-29 years in both COS and FET showed significantly lower levels in the youngest women. CONCLUSION(S): The initial hCG rise was influenced by the age of the woman, with an identical pattern for hCG values in COS and FET treatments. In COS, the competent blastocyst had a reduced stage with increasing women's age.

RUBIC (ReproUnion Biobank and Infertility Cohort): A binational clinical foundation to study risk factors, life course, and treatment of infertility and infertility-related morbidity.

BACKGROUND: Infertility affects 15%-25% of all couples during their reproductive life span. It is a significant societal and public health problem with potential psychological, social, and economic consequences. Furthermore, infertility has been linked to adverse long-term health outcomes. Despite the advanced diagnostic and therapeutic techniques available, approximately 30% of infertile couples do not obtain a live birth after fertility treatment. For these couples, there are no further options to increase their chances of a successful pregnancy and live birth. OBJECTIVES: Three overall questions will be studied: (1) What are the risk factors and natural life courses of infertility, early embryonic loss, and adverse pregnancy outcomes? (2) Can we develop new diagnostic and prognostic biomarkers for fecundity and treatment success? And (3) what are the health characteristics of women and men in infertile couples at the time of fertility treatment and during long-term follow-up? MATERIAL AND METHODS: ReproUnion Biobank and Infertility Cohort (RUBIC) is established as an add-on to the routine fertility management at Copenhagen University Hospital Departments in the Capital Region of Denmark and Reproductive Medicine Centre at Skåne University Hospital in Sweden. The aim is to include a total of 5000 couples equally distributed between Denmark and Sweden. The first patients were enrolled in June 2020. All eligible infertile couples are prospectively asked to participate in the project. Participants complete an extensive questionnaire and undergo a physical examination and collection of biospecimens (blood, urine, hair, saliva, rectal swabs, feces, semen, endometrial biopsies, and vaginal swabs). After the cohort is established, the couples will be linked to the Danish and Swedish national registers to obtain information on parental, perinatal, childhood, and adult life histories, including disease and medication history. This will enable us to understand the causes of infertility and identify novel therapeutic options for this important societal problem.