Mitochondrial DNA variants segregate during human preimplantation development into genetically different cell lineages that are maintained postnatally.

Humans present remarkable diversity in their mitochondrial DNA (mtDNA) in terms of variants across individuals as well as across tissues and even cells within one person. We have investigated the timing of the first appearance of this variant-driven mosaicism. For this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 embryos present blastomeres that carry variants only detected in that cell, showing that mtDNA mosaicism arises very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across different samples. Recurring variants had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants unique to one oocyte or single embryonic cell. These differences were maintained through development, suggesting that the mtDNA mosaicism arising in the embryo is maintained into adulthood. We observed a decline in potentially pathogenic variants between day 3 and day 5 of development, suggesting early selection. We propose a model in which closely clustered mitochondria carrying specific mtDNA variants in the ooplasm are asymmetrically distributed throughout the cell divisions of the preimplantation embryo, resulting in the earliest form of mtDNA mosaicism in human development.

Clinical pregnancy rates after blastocyst culture at a stable temperature of 36.6°C versus 37.1°C: a prospective randomized controlled trial.

STUDY QUESTION: Is there a difference in clinical pregnancy rates (CPRs) in good prognosis patients after single embryo transfer (SET) on Day 5, in case of stable culture at 36.6°C or 37.1°C? SUMMARY ANSWER: CPR (with heartbeat at 7 weeks) after blastocyst transfer do not differ after culturing at 36.6°C or 37.1°C. WHAT IS KNOWN ALREADY: Since the beginning of IVF, embryo culture has been performed at 37.0°C; however, the optimal culture temperature remains unknown. Changes in incubator types have led to significant improvements in temperature control. Stable temperature control, i.e. with temperature differences of max. 0.1°C between chambers, is possible in some incubators. A previous prospective pilot study showed that embryo development on Day 5/6 was not affected when embryos were cultured at a stable temperature of 36.6°C or 37.1°C, but culture at 37.1°C resulted in an increased CPR when compared to culture at 36.6°C (74.2% vs 46.4%). STUDY DESIGN, SIZE, DURATION: A prospective randomized controlled trial was performed in a tertiary fertility centre between February 2017 and November 26, 2022. A sample size of 89/89 patients with fresh single embryo transfer (SET) was required to achieve 80% power to detect a difference of 0.22 between group proportions (0.43-0.65) at a significance level of 0.05 using a two-sided z-test with continuity correction. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were recruited on the day of oocyte retrieval based on inclusion criteria with final randomization after denudation once six mature oocytes were present. The primary endpoint was CPR (heartbeat at 7 weeks); secondary endpoints were fertilization rate, blastocyst development, biochemical pregnancy rate, live birth rate (LBR), and cumulative live birth rate (CLBR). MAIN RESULTS AND THE ROLE OF CHANCE: A total of 304 patients were eligible for the study; of these 268 signed the consent, 234 (intention-to-treat) were randomized and 181 (per-protocol) received a SET on Day 5: 90 received culture at 36.6°C and 91 at 37.1°C. Patients were on average 32.4 ± 3.5 versus 32.5 ± 4.2 years old, respectively. No differences were observed in embryological outcomes per cycle between culture at 36.6°C versus 37.1°C: 12.0 ± 3.8 vs 12.1 ± 3.8 COCs retrieved (P = 0.88), 10.0 ± 3.1 versus 9.9 ± 2.9 mature oocytes inseminated (P = 0.68), with a maturation rate of 84.2% (901/1083) versus 83.5% (898/1104) (P = 0.87); and 8.0 ± 3.1 versus 7.9 ± 2.7 normally fertilized oocytes with a fertilization rate of 79.7% (720/901) vs 80.5% (718/898) (P = 0.96), respectively. On average 1.5 ± 1.7 versus 1.4 ± 1.9 (P = 0.25) and 1.1 ± 1.1 versus 0.9 ± 1.0 (P = 0.45) supernumerary blastocysts were vitrified on Day 5 and Day 6, respectively. The utilization rate per fertilized oocyte was 46.1% vs 41.5% (P = 0.14). A SET was performed for 181 patients, leading to a biochemical pregnancy rate of 72.2% (65/90) versus 62.7% (57/91) (P = 0.17), respectively. The CPR per fresh transfer cycle was 51.1% (46/90) versus 48.4% (44/91) [OR (95% CI) 1.11 (0.59-2.08), P = 0.710]. To date, a CLBR of 73.3% (66/90) versus 67.0% (61/91) (P = 0.354) has been observed, respectively. In each group, seven patients without live birth have remaining blastocysts frozen. The CPR for the intention-to-treat groups were 38.3% vs 38.6% [OR (95% CI) 0.98 (0.56-1.73), P = 0.967], respectively, for culture at 36.6°C versus 37.1°C. LIMITATIONS, REASONS FOR CAUTION: Only selected patients with expected good prognosis were eligible for the study. WIDER IMPLICATIONS OF THE FINDINGS: Embryos tend to tolerate small changes in temperature deviations during culture to the blastocyst stage, as demonstrated by their similar implantation potential at two slightly different temperatures. STUDY FUNDING/COMPETING INTEREST(S): There is no funding or conflicts of interest to declare. TRIAL REGISTRATION NUMBER: NCT03548532. TRIAL REGISTRATION DATE: 23 October 2017. DATE OF FIRST PATIENT'S ENROLMENT: 10 November 2017.

Circadian serum progesterone variations on the day of frozen embryo transfer in artificially prepared cycles.

RESEARCH QUESTION: What is the intra-day variation of serum progesterone related to vaginal progesterone administration on the day of frozen embryo transfer (FET) in an artificial cycle? DESIGN: A prospective cohort study was conducted including 22 patients undergoing a single blastocyst artificial cycle (AC)-FET from August to December 2022. Endometrial preparation was achieved by administering oestradiol valerate (2 mg three times daily) and consecutively micronized vaginal progesterone (MVP; 400 mg twice daily). A blastocyst FET was performed on the 6th day of MVP administration. Serum progesterone concentrations were measured on the day of transfer at 08:00, 12:00, 16:00 and 20:00 hours. The first and last blood samples were collected just before MVP was administered. RESULTS: The mean age and body mass index of the study population were 33.95 ± 3.98 years and 23.10 ± 1.95 kg/m2. The mean P-values at 08:00, 12:00, 16:00 and 20:00 hours were 11.72 ± 4.99, 13.59 ± 6.33, 10.23 ± 3.81 and 9.28 ± 3.09 ng/ml, respectively. A significant decline, of 2.41 ng/ml (95% confidence interval 0.81-4.00), was found between the first and last progesterone measurements. CONCLUSION: A statistically significant intra-day variation of serum progesterone concentrations on the day of FET in artificially prepared cycles was observed. This highlights the importance of a standardized procedure for the timing of progesterone measurement on the day of AC-FET. Of note, the study results are applicable only to women using MVP for luteal phase support; therefore it is necessary to confirm its validity in comparison with the different existing administration routes of progesterone.

Early versus late follicular phase ovarian stimulation: a randomized controlled trial.

RESEARCH QUESTION: Is late follicular phase stimulation as efficient as early follicular phase stimulation in a gonadotrophin-releasing hormone (GnRH) antagonist protocol in oocyte donors in terms of the number of oocytes. DESIGN: In this open label, phase 3, non-inferiority, randomized controlled trial using a two-arm design with a 1:1 allocation ratio, 84 oocyte donors were allocated to the early follicular start group (control group, n = 41) or the late follicular start group (study group, n = 43). In the control group, women followed a fixed GnRH antagonist protocol with recombinant FSH (r-FSH) 225 IU. In the study group, r-FSH 225 IU was initiated in the late follicular phase. The primary outcome was the number of oocytes. The secondary outcomes were the number of mature oocytes, consumption of gonadotrophins and GnRH antagonist, and cost of medication. RESULTS: The number of oocytes did not differ between the control group and the study group (intent-to-treat analysis 15.5 ± 11.0 versus 14.0 ± 10.7, P = 0.52; per-protocol analysis 18.2 ± 9.7 versus 18.8 ± 7.8, P = 0.62). In addition, the number of mature oocytes did not differ between the groups (14.1 ± 8.1 versus 12.7 ± 8.5, P = 0.48). The duration of stimulation was shorter in the control group (10.0 ± 1.4 versus 10.9 ± 1.5 days, P = 0.01). The total amount of r-FSH used was lower in the control group (2240.7 ± 313.9 IU versus 2453.9 ± 330.1 IU, P = 0.008). A GnRH antagonist was used for approximately 6 days in the control group, while a GnRH antagonist was only prescribed for one woman in the study group (6.0 ± 1.4 days versus 0.13±0.7 days, P < 0.001). There was a significant difference in the cost of medication per cycle between the groups (1147.9 ± 182.8€ in control group versus 979.9 ± 129.0€ in study group, P < 0.001). CONCLUSIONS: Late follicular phase stimulation is as efficient as early follicular phase stimulation in terms of the number of oocytes.

IVF with reception of oocytes from partner in lesbian couples: a systematic review and SWOT analysis.

The growing utilization of assisted reproductive technology (ART) by the LGBTQ+ community, especially among lesbian couples, challenges societal norms and promotes inclusivity. The reception of oocytes from partner (ROPA) technique enables both female partners to have a biological connection to their child. A systematic review was conducted of the literature on ROPA IVF to provide the latest data and a SWOT analysis was subsequently performed to understand the strengths, weaknesses, opportunities and threats associated with ROPA IVF. Publications from 2000 to 2023 with relevant keywords were reviewed and 16 records were included. Five studies provided clinical information on couples who used ROPA IVF. ROPA IVF provides a unique opportunity for a biological connection between the child and both female partners and addresses concerns related to oocyte donation and anonymity. Weaknesses include limited cost-effectiveness data and unresolved practical implications. Opportunities lie in involving both partners in parenthood, advancing ART success rates and mitigating risks. Threats encompass increased pregnancy complications, ethical concerns, insufficient safety data, legal or cultural barriers, and emotional stress. In conclusion, ROPA IVF offers a promising solution for lesbian couples seeking to create a family in which both partners want to establish a biological connection with their child.

Optimized sperm selection: a highly efficient device for the isolation of progressive motile sperm with low DNA fragmentation index.

PURPOSE: To identify the sperm preparation procedure that selects the best sperm population for medically assisted reproduction. METHODS: Prospective observational study comparing the effect of four different sperm selection procedures on various semen parameters. Unused raw semen after routine diagnostic analysis was split in four fractions and processed by four different methods: (1) density gradient centrifugation (DGC), (2) sperm wash (SW), (3) DGC followed by magnetic activated cell sorting (MACS), and (4) using a sperm separation device (SSD). Each fraction was analyzed for progressive motility, morphology, acrosome index (AI), and DNA fragmentation index (DFI). RESULTS: With DGC as standard of care in intraclass correlation coefficient analysis, only SSD was in strong disagreement regarding progressive motility and DFI [0.26, 95%CI (- 0.2, 0.58), and 0.17, 95%CI (- 0.19, 0.45), respectively]. When controlling for abstinence duration, DFI was significantly lower after both MACS and SSD compared to DGC [- 0.27%, 95%CI (- 0.47, - 0.06), p = 0.01, and - 0.6%, 95%CI (- 0.80, - 0.41), p < 0.001, respectively]. Further comparisons between SSD and MACS indicate significantly less apoptotic cells [Median (IQR) 4 (5), 95%CI (4.1, - 6.8) vs Median (IQR) 5 (8), 95%CI (4.9, - 9.2), p < 0.001, respectively] and dead cells [Median (IQR) 9.5 (23.3), 95%CI (13.2, - 22.4) vs Median (IQR) 22 (28), 95%CI (23.1, - 36.8), p < 0.001, respectively] in the SSD group. CONCLUSION: The selection of a population of highly motile spermatozoa with less damaged DNA from unprocessed semen is ideally performed with SSD. Question remains whether this method improves the embryological outcomes in the IVF laboratory.

Electronic witnessing in the medically assisted reproduction laboratory: insights and considerations after 10 years of use.

STUDY QUESTION: What have we learnt after 10 years of electronic witnessing? SUMMARY ANSWER: When applied correctly, an electronic witnessing system can replace manual witnessing in the medically assisted reproduction lab to prevent sample mix-up. WHAT IS KNOWN ALREADY: Electronic witnessing systems have been implemented to improve the correct identification, processing, and traceability of biological materials. When non-matching samples are simultaneously present in a single workstation, a mismatch event is generated to prevent sample mix-up. STUDY DESIGN, SIZE, DURATION: This evaluation investigates the mismatch and administrator assign rate over a 10-year period (March 2011-December 2021) with the use of an electronic witnessing system. Radiofrequency identification tags and barcodes were used for patient and sample identification. Since 2011, IVF and ICSI cycles and frozen embryo transfer cycles (FET) were included; IUIs cycles were included since 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: The total number of tags and witnessing points were recorded. Witnessing points in a particular electronic witnessing system represent all the actions that have been performed from gamete collection through embryo production, to cryopreservation and transfer. Mismatches and administrator assigns were collected and stratified per procedure (sperm preparation, oocyte retrieval, IVF/ICSI, cleavage stage embryo or blastocyst embryo biopsy, vitrification and warming, embryo transfer, medium changeover, and IUI). Critical mismatches (such as mislabelling or non-matching samples within one work area) and critical administrator assigns (such as samples not identified by the electronic witnessing system and unconfirmed witnessing points) were selected. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 109 655 cycles were included: 53 023 IVF/ICSI, 36 347 FET, and 20 285 IUI cycles. The 724 096 used tags, led to a total of 849 650 witnessing points. The overall mismatch rate was 0.251% (2132/849 650) per witnessing point and 1.944% per cycle. In total, 144 critical mismatches occurred over the different procedures. The yearly mean critical mismatch rate was 0.017 ± 0.007% per witnessing point and 0.129 ± 0.052% per cycle. The overall administrator assign rate was 0.111% (940/849 650) per witnessing point and 0.857% per cycle, including 320 critical administrator assigns. The yearly mean critical administrator assign rate was 0.039 ± 0.010% per witnessing point and 0.301 ± 0.069% per cycle. Overall mismatch and administrator assign rates remained fairly stable during the evaluated time period. Sperm preparation and IVF/ICSI were the procedures most prone to critical mismatch and administrator assigns. LIMITATIONS, REASONS FOR CAUTION: The procedures and methods of integration of an electronic witnessing system may vary from one laboratory to another and result in differences in the potential risks related to sample identification. Individual embryos cannot (yet) be identified by such a system; this makes extra manual witnessing indispensable at certain critical steps where potential errors are not recorded. The electronic witnessing system still needs to be used in combination with manual labelling of both the bottom and lid of dishes and tubes to guarantee correct assignment in case of malfunction or incorrect use of radiofrequency identification tags. WIDER IMPLICATIONS OF THE FINDINGS: Electronic witnessing is considered to be the ultimate tool to safeguard correct identification of gametes and embryos. But this is only possible when used correctly, and proper training and attention of the staff is required. It may also induce new risks, i.e. blind witnessing of samples by the operator. STUDY FUNDING/COMPETING INTEREST(S): No funding was either sought or obtained for this study. J.S. presents webinars on RIW for CooperSurgical. The remaining authors have nothing to declare. TRIAL REGISTRATION NUMBER: N/A.

Selecting active matter according to motility in an acoustofluidic setup: self-propelled particles and sperm cells.

Active systems - including sperm cells, living organisms like bacteria, fish, birds, or active soft matter systems like synthetic "microswimmers" - are characterized by motility, i.e., the ability to propel using their own "engine". Motility is the key feature that distinguishes active systems from passive or externally driven systems. In a large ensemble, motility of individual species can vary in a wide range. Selecting active species according to their motility represents an exciting and challenging problem. We propose a new method for selecting active species based on their motility using an acoustofluidic setup where highly motile species escape from the acoustic trap. This is demonstrated in simulations and in experiments with self-propelled Janus particles and human sperm. The immediate application of this method is selecting highly motile sperm for medically assisted reproduction (MAR). Due to the tunable acoustic trap, the proposed method is more flexible than the existing passive microfluidic methods. The proposed selection method based on motility can also be applied to other active systems that require selecting highly motile species or removing immotile species.

Female parental consanguinity is associated with a reduced ovarian reserve.

RESEARCH QUESTION: Is parental consanguinity associated with a reduced ovarian reserve in women from the Arabian Peninsula, comparing anti-Müllerian hormone (AMH) and antral follicle count (AFC)? DESIGN: Retrospective large-scale observational study including 2482 women from the Arabian Peninsula, aged 19-49 years, who had their serum AMH and AFC measured as part of their fertility assessment, from May 2015 to November 2019. Consanguinity was defined as women whose parents were first-degree or second-degree cousins. Serum AMH was measured for all participants. RESULTS: A total of 2198 women were included: 605 in the consanguine group (27.53%), 1593 (72.47%) in the non-consanguine group. There were no significant differences between groups in terms of body mass index, years of infertility or smoking status. Women from the consanguine group were significantly younger (mean age 33.74 ± 6.64 years) compared with the non-consanguine group (mean age 34.78 ± 6.64 years, P < 0.0001). Median AMH and AFC for the consanguine group were 1.90 ng/ml (min-max: 0.01-23.8) and 11 (0-80), respectively, and for the non-consanguine group 1.84 ng/ml (min-max: 0.01-23.0) and 11 (0-60), respectively. AMH and AFC exhibit an age-dependent decline. As both parameters are age-dependent, the multivariate analysis showed that women from the consanguine group presented significantly lower AMH (coefficient of variation [CV] -0.07 ± 0.03, P = 0.036) and AFC (CV -0.16 ± 0.06, P = 0.003) compared with non-consanguine women, and the highest differences were found for women below 35 years of age (AMH median [min-max]: 2.82 ng/ml (0.01-23.80) versus 2.92 ng/ml (0.01-23.00); P = 0.035; AFC median [min-max]: 15 (0-80) versus 14 (0-80); P = 0.001). CONCLUSION: The adjusted analysis by age indicates that female parental consanguinity is associated with reduced ovarian reserve in the studied population. Clinical evaluation should include extensive family history and subsequent counselling of the affected couples.

Day 5 vs day 6 single euploid blastocyst frozen embryo transfers: which variables do have an impact on the clinical pregnancy rates?

OBJECTIVE: To determine which variables affect most the clinical pregnancy rate with positive fetal heartbeat (CPR FHB+) when frozen embryo transfer (FET) cycles are performed with day 5 (D5) or day 6 (D6) euploid blastocysts. Design and method A single center retrospective study was performed from March 2017 till February 2021 including all single FET cycles with euploid D5 or D6 blastocysts and transferred in natural cycles (NC) or hormone replacement therapy (HRT) cycles. Trophectoderm (TE) and inner cell mass (ICM) qualities were recorded before biopsy. RESULTS: A total of 1102 FET cycles were included, 678 with D5 and 424 with D6 blastocysts. Pregnancy rate (PR), clinical PR (CPR), and CPR FHB+ were significantly higher with D5 blastocysts (PR: 70.7% vs 62.0%, OR = 0.68 [0.53-0.89], p = 0.004; CPR: 63.7% vs 54.2%, OR = 0.68 [0.52-0.96], p = 0.002 and CPR FHB+: 57.8% vs 49.8%, OR = 0.72 [0.53-0.96], p = 0.011). However, miscarriage rate (12.5% vs 11.4%, OR = 0.78 [0.48-1.26], p = 0.311) did not differ. From a multivariate logistic regression model, endometrial thickness (OR = 1.11 [1.01-1.22], p = 0.028), patient's age (OR = 1.03 [1.00-1.05], p = 0.021), BMI (OR = 0.97 [0.94-0.99], p = 0.023), and ICM grade C (OR = 0.23 [0.13-0.43], p < 0.001) were significant in predicting CPR FHB+. CONCLUSION: Although clinical outcomes are higher with D5 blastocysts, CPR FHB+ is more affected by endometrial thickness, patient age, BMI, and ICM grade C rather than biopsy day or endometrial preparation protocol.