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.

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation.

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) is still the only option to preserve fertility in women who need to start gonadotoxic treatment urgently or in prepubertal children. During ovarian cortex preparation for cryopreservation, medullar tissue is removed. Growing antral follicles reside at the border of the cortex-medullar interface of the ovary and are broken during this process, releasing their cumulus-oocyte complex (COC). By thoroughly inspecting the medium and fragmented medullar tissue, these immature cumulus-oocyte complexes can be identified without interfering with the OTC procedure. The ovarian tissue-derived immature oocytes can be successfully matured in vitro, creating an additional source of gametes for fertility preservation. If OTC is performed within or near a medical assisted reproduction laboratory, all necessary in vitro maturation (IVM) and oocyte vitrification tools can be at hand. Furthermore, upon remission and child wish, the patient has multiple options for fertility restoration: ovarian tissue transplantation or embryo transfer after the insemination of vitrified/warmed oocytes. Hence, ovarian tissue oocyte-in vitro maturation (OTO-IVM) can be a valuable adjunct fertility preservation technique.

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.

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.

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.

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.

The Ratio of Serum Progesterone (P4) to the Number of Follicles (P4/follicle) is a More Objective Parameter for Euploidy Rate as Compared to Systemic Progesterone Levels.

Does the late follicular phase progesterone (P4) and the P4-to-follicle-ratio affect the ploidy state of the biopsied embryos? A retrospective observational study conducted at ART Fertility Clinics Abu Dhabi and Muscat, including all stimulation cycles performed between January 2015 and December 2019. In total, 975 cycles were considered for this study. Inclusion criteria were ovarian stimulation due to primary/secondary infertility, patient's age between 18 and 45 years, ICSI as fertilization method, and patients undergoing preimplantation genetic testing for aneuploidies (PGT-A). Patients with testicular sperm extraction (TESE) and warmed oocytes were excluded. Our results have shown that progesterone had no effect on the euploid rate (p = 0.371). However, when adding the ratio of P4 to the number of follicles that were bigger than 10 mm in the last scan, a negative effect on the euploid rate (p < 0.05) was observed. This study was able to show that the use of only P4 is unable to predict ploidy outcomes. However, by including the number of follicles > 10 mm, a clear association was observed between P4/Foll ratio and euploid rate per cycle. The use of both parameters could aid clinicians in their decision to trigger a patient or continue stimulation. Further prospective studies are warranted to confirm those results.

Marginal differences in preimplantation morphokinetics between conventional IVF and ICSI in patients with preimplantation genetic testing for aneuploidy (PGT-A): A sibling oocyte study.

OBJECTIVE: This study aimed to analyze the morphokinetic behaviour between conventional IVF and ICSI, in cycles with preimplantation genetic testing for aneuploidies (PGT-A). MATERIALS: A randomized controlled trial (NCT03708991) was conducted in a private fertility center. Thirty couples with non-male factor infertility were recruited between November 2018 and April 2019. A total of 568 sibling cumulus oocyte complexes were randomly inseminated with conventional IVF and ICSI and cultured in an Embryoscope time-lapse system. The morphokinetic behaviour of IVF/ICSI sibling oocytes was analysed as primary endpoint. As secondary endpoints, morphokinetic parameters that predict blastocysts that will be biopsied, the day of biopsy, gender and euploid outcome was assessed. RESULTS: When comparing IVF to ICSI, only the time to reach the 2-cell stage (t2) was significantly delayed for IVF embryos: OR: 1.282 [1.020-1.612], p = 0.033. After standardizing for tPNf (ct parameters), only Blast(tStartBlastulation-t2) remained significant: OR: 0.803 [0.648-0.994], p = 0.044. For the analysis of zygotes that will be biopsied on day 5/6 versus zygotes without biopsy, only early morphokinetic parameters were considered. All parameters were different in the multivariate model: ct2: OR: 0.840 [0.709-0.996], p = 0.045; ct6: OR: 0.943 [0.890-0.998], p = 0.043; cc2(t3-t2): OR: 1.148 [1.044-1.263], p = 0.004; cc3(t5-t3): OR: 1.177 [1.107-1.251], p<0.0001. When comparing the development between blastocysts biopsied on day 5 versus day 6, only three morphokinetic parameters were significant: cc2(t3-t2): OR: 1.394 [1.010-1.926], p = 0.044; ctBlastocyst: OR: 0.613 [0.489-0.768], p<0.0001 and ctExpandedBlastocyst: OR: 0.913 [0.868-0.960], p = 0.0004. Multivariate analysis of gender and ploidy did not reveal differences in morphokinetic behaviour. CONCLUSION: Minor morphokinetic differences are observed between IVF and ICSI. Early in the development, distinct cleavage patterns are observed between embryos that will be biopsied or not.

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.

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.

Ethnic and Sociocultural Differences in Ovarian Reserve: Age-Specific Anti-Müllerian Hormone Values and Antral Follicle Count for Women of the Arabian Peninsula.

Background: Anti-Müllerian hormone (AMH) and antral follicle count (AFC) age-specific reference values form the basis of infertility treatments, yet they were based upon studies performed primarily on Caucasian populations. However, they may vary across different age-matched ethnic populations. This study aimed to describe age-specific serum AMH and AFC for women native to the Arabian Peninsula. Methods: A retrospective large-scale study was performed including 2,495 women, aged 19 to 50 years, native to the Arabian Peninsula. AMH and AFC were measured as part of their fertility assessment at tertiary-care fertility centres. Age-specific values and nomograms were calculated. Results: 2,495 women were evaluated. Mean, standard deviation and median values were calculated for AMH and AFC by 1-year and 5-years intervals. Median age was 34.81 years, median AMH was 1.76ng/ml and median AFC was 11. From the total group, 40.60% presented with AMH levels below 1.3ng/mL. For women <45 years old, the decrease in AFC was between -0.6/-0.8 per year. Up to 36 years old, the decrease of AMH was 0.1ng/ml. However, from 36 to 40 years old, an accelerated decline of 0.23ng/ml yearly was noted. In keeping with local customs, 71.23% of women wore the hijab and 25.76% the niqab. AMH and AFC were significantly lower for niqab group compared with hijab group (p=0.02 and p=0.04, respectively). Conclusion: This is to-date the largest data set on age-specific AMH and AFC values in women from the Arabian Peninsula aiming to increase clinical awareness of the ovarian reserve in this population.

The position of the euploid blastocyst in the uterine cavity influences implantation.

RESEARCH QUESTION: Does the position of the euploid blastocyst in the uterine cavity upon transfer, measured as distance in millimetres (mm) from the fundus (DFF) to the air bubble, influence implantation potential? DESIGN: A total of 507 single/double euploid frozen embryo transfer (FET) cycles at blastocyst stage were included retrospectively between March 2017 and November 2018 at a single centre. The patients were on average 33.3 years old. The FET were performed in natural cycles (n = 151) or hormone replacement therapy cycles (n = 356). RESULTS: Of the 507 transfers, 370 (73.0%) resulted in a pregnancy, defined as human chorionic gonadotrophin concentration over 15 mIU/ml, and 341 (67.3%) in a clinical pregnancy, with an implantation rate of 62.0% and ongoing pregnancy rate of 59.6% (302/507). When comparing the number of embryos transferred, the pregnancy rate, clinical pregnancy rate and ongoing pregnancy rate were significantly higher after double-embryo transfer (DET) (P = 0.002: P < 0.001 and P = 0.002). The quality of the blastocyst in the single-embryo transfer group had a positive effect on the pregnancy rate (A versus B, P = 0.016; A versus C, P = 0.003) and clinical pregnancy rate (A versus C, P = 0.013). After performing a multivariate logistic regression analysis to consider the effect of all explanatory variables, a negative effect between DFF and pregnancy (P = 0.001), clinical pregnancy (P = 0.001) and ongoing pregnancy (P = 0.030) was found. When all variables remained constant, an increase of 1 mm of DFF changed the odds of pregnancy by 0.882, of clinical pregnancy by 0.891 and of ongoing pregnancy by 0.925. No significant effect of DFF was found on the miscarriage outcome (P = 0.089). CONCLUSIONS: The depth of blastocyst replacement inside the uterine cavity may influence the pregnancy, clinical pregnancy and ongoing pregnancy rates and should be considered as an important factor to improve the success of IVF cycles.

Different CO2 settings (6.0% vs 7.0%) do have an impact on extracellular pH of culture medium (pHe) and euploidy rates rather than on blastocyst development: a sibling oocyte study.

OBJECTIVE: To determine whether euploidy rates and blastocyst development differ in a continuous culture medium under different CO2 concentrations. DESIGN AND METHOD: A single-center retrospective study was performed from July 2018 to October 2019 including 44 fresh cycles with at least four fresh mature oocytes (MII) without severe male factor infertility. Sibling MII were injected and cultured in Global®Total®LP under 6.0% (pHe = 7.374 ± 0.014) or 7.0% (pHe = 7.300 ± 0.013) CO2, 5.0% O2, and 89.0% or 88.0% N2. Analyzed variables were normally fertilized oocytes (2PN), cleavage rate, blastulation rate on day 5/2PN, usable blastocyst (blastocysts biopsied/2PN), and euploidy rates. Blastocyst's trophectoderm biopsy was performed on day 5, 6, or 7 for genetic testing and mitochondrial DNA (mtDNA) quantification by next-generation sequencing. RESULTS: Women's mean age was 33.0 ± 6.6 years old. From a total of 604 MII, no differences were found in normal fertilization and cleavage rates on day 3 between 6.0 and 7.0% CO2 (72.3% vs 67.1%, p = 0.169 and 96.6% vs 96.3%, p = 0.897, respectively). Blastulation rate on day 5/2PN was comparable between 6.0 and 7.0% CO2 (68.1% vs 64.2%, p = 0.409). Although usable blastocyst rate was not different (54.3% vs 55.3%, p = 0.922), total euploidy rates differed significantly (58.7% vs 42.8%, p = 0.016) between 6.0% and 7.0% CO2, respectively. The mean blastocyst mtDNA content was significantly lower in 6.0% CO2 (30.4 ± 9.1 vs 32.9 ± 10.3, p = 0.037). CONCLUSION: Blastocyst development is not affected when embryos are cultured in vitro at 6.0% or 7.0% CO2, while euploidy rates are significantly decreased at a higher CO2 concentration, therefore at a lower pHe.

Euploidy rates are not affected when embryos are cultured in a continuous (CCM) or sequential culture medium (SCM): a sibling oocyte study.

PURPOSE: To determine if euploidy rates and embryo development differ when blastocysts are cultured in CCM or SCM. METHOD: A single-center retrospective observational study was performed from September 2018 to March 2019. Patients [23-46 years] with at least four fresh mature oocytes (MII) without severe male factor infertility were included. Sibling MII were injected and cultured in Global®Total®LP (CCM) or Sage Quinn's Advantage® Cleavage and Blastocyst media (SCM) under 6% CO2, 5% O2, and 89% N2. Fertilization, cleavage, day (D) 5 blastulation, usable blastocyst (blastocysts biopsied/normally fertilized oocytes), and euploidy rates were recorded. Blastocysts were graded prior to trophectoderm (TE) biopsy on D5, 6, or 7 for genetic testing and mitochondrial DNA (mtDNA) quantification. RESULTS: According to clinical practice, 1452 MII were randomly distributed: 751 in CCM and 701 in SCM. No differences were observed in fertilization and cleavages rates for CCM and SCM (77.4% vs 75.5%, p = 0.429 and 97.6% vs 99.1%, p = 0.094, respectively). Blastulation rate on D5 was higher in CCM (70.6% vs 62.2, p = 0.009); however, usable blastocyst rates were comparable (CCM: 58.3% vs SCM: 56.7%, p = 0.625). From a Poisson regression model adjusted for confounding factors, euploidy rates were not different between media (aOR = 1.18, [0.94-1.48], p = 0.157). Euploid blastocyst's mtDNA values were similar (CCM: 32.2, [30.5, 34.1] and SCM: 33.5, [31.8, 35.2], p = 0.345) and top-quality blastocysts (AA/BA) were increased in SCM (OR=1.04, [1.00-1.09], p = 0.037). CONCLUSION: Under controlled in vitro conditions, euploidy rates and embryo development are comparable when embryos are cultured in CCM or SCM.

Does blastocyst mitochondrial DNA content affect miscarriage rate in patients undergoing single euploid frozen embryo transfer?

PURPOSE: To determine whether the blastocyst mitochondrial DNA (mtDNA) content is related to the miscarriage rate in patients undergoing single euploid frozen embryo transfer (SEFET). METHODS: A total of 355 single euploid frozen embryo transfer cycles were studied retrospectively between April 2017 and December 2018. A trophectoderm biopsy was performed on day 5/6 blastocysts. Post next-generation sequencing (NGS), the mtDNA content was calculated as the ratio of mitochondrial DNA over nuclear DNA, and the association between blastocyst mtDNA content and miscarriage rate was evaluated. RESULT(S): Three hundred fifty-five euploid blastocysts were selected for SEFET in 314 patients with an average age of 33.7 ± 5.6 years; 255 were biopsied on day 5 (71.8%) and 100 on day 6 (28.2%). Frozen embryo transfer (FET) was performed either in a hormone replacement therapy (HRT) cycle (71.8%; n = 255) or in a natural cycle (NC) (28.2%; n = 100). A pregnancy rate of 66.2% (235/355) was obtained with clinical pregnancy and miscarriage rates of 52.4% (n = 186) and 5.6% (n = 20), respectively. There was no significant difference neither between the blastocyst mtDNA content of pregnant and nonpregnant patients (27.7 ± 9.2 vs. 29.4 ± 8.6, P = 0.095) nor between patients with a clinical pregnancy and miscarriage (30.5 ± 9.3 vs. 27.3 ± 9.2, P = 0.136). Multivariate logistic regression analysis showed the same nonsignificant relationship, except for the miscarriage rate and BMI (OR 1.149, 95% CI 1.03-1.28; P = 0.012). CONCLUSION(S): Mitochondrial DNA content is unable to predict the miscarriage of implanted human euploid blastocysts.

Anti-Müllerian hormone is an independent marker for oocyte survival after vitrification.

RESEARCH QUESTION: This study explored the relationship between anti-Müllerian hormone (AMH) and oocyte survival after vitrification. The association between AMH and blastocyst formation after oocyte vitrification was also assessed. DESIGN: A retrospective observational analysis was performed in a private IVF centre. A total of 4507 metaphase-II warmed oocytes were included from 450 couples, predominantly of Arab ethnicity. Between August 2015 and August 2018, couples underwent 484 intracytoplasmic sperm injection (ICSI) treatments using vitrified-warmed oocytes. RESULTS: Patients' median age ± SD was 36.2 ± 6.1 years, AMH concentration 2.6 ± 3.4 ng/ml and body mass index (BMI) 26.5 ± 4.6 kg/m2. The oocyte survival rate after vitrification was 87.37 ± 20.42%. AMH concentration showed a significant correlation (Kendall's tau 0.087, P = 0.0079) with oocyte survival rate independent of oocyte yield. Correlation was significant (odds ratio 1.041, 95% confidence interval 1.007-1.077, P = 0.018) when a multivariant model was applied that included AMH, age and BMI. The receiver operating characteristic curve showed an AMH cut-off value of 1.09 ng/ml that could obtain at least a 70% survival rate, with an area under the curve of 0.669. Regarding embryo development in ICSI cycles including fresh and warmed oocytes for the same patient, blastocyst formation rate was higher in fresh compared with warmed oocytes (P < 0.001). In this subgroup no significant correlation was seen between fertilization or blastocyst rate and AMH concentration. CONCLUSIONS: AMH concentration showed a significant correlation with oocyte survival. Blastocyst formation was significantly lower after oocyte vitrification, but no correlation was found with AMH. Clinicians should carefully evaluate oocyte vitrification for patients with AMH below 1.09 ng/ml and consider embryo accumulation for these patients in preference to oocyte accumulation.

Impact of Serum Estradiol Levels Prior to Progesterone Administration in Artificially Prepared Frozen Embryo Transfer Cycles.

Background: The need for endocrine monitoring in artificial cycles for frozen embryo transfer (FET) remains unclear and, more specifically, the value of the late-proliferative phase serum estradiol (E2) levels is with conflicting evidence in current literature. Objective: To investigate whether artificial FET cycles require endocrine monitoring for the serum E2 level prior to initiation of exogenous progesterone administration after an endometrial thickness of 6.5 mm has been reached. Design: One thousand two hundred and twenty-two (n = 1,222) artificial FETs performed in a tertiary center between 2010 and 2015 were subdivided into 3 groups according to the following late-proliferative serum E2 level percentiles: ≤p10 (E2 ≤144 pg/ml; n = 124), p11-p90 (E2 from 145 to 438 pg/ml; n = 977) and >p90 (E2 >439 pg/ml; n = 121). A mixed-effects multilevel multivariable regression analysis was performed to assess the potential effect of the late-proliferative E2 level on the live birth rate (LBR). Results: The level of late-proliferative circulating E2 showed no significant difference in terms of LBR after FET. Specifically, the multivariable regression model demonstrated a LBR of 19.5% for the p11-p90 reference group, compared to 24.4% for the ≤p10 (p = 0.251) and 19.5% for the >p90 group (p = 0.989). Conclusion: In this large retrospective dataset, no association was observed between late-proliferative phase serum E2 levels and LBR following FET in artificially prepared cycles. Although, caution is warranted due to the retrospective nature of the analysis and the potential for unmeasured confounding, we argue that monitoring of the late-proliferative serum E2 levels and using them to guide clinical decision-making (e.g., medication step-up, cycle prolongation or cancelation) may be of questionable value.

Blastocyst mitochondrial DNA (mtDNA) is not affected by oocyte vitrification: a sibling oocyte study.

PURPOSE: To evaluate whether mtDNA content at the blastocyst stage differs between embryos derived from fresh or vitrified sibling oocytes. MATERIAL AND METHODS: A retrospective analysis was performed between March 2017 and September 2018, including 504 blastocysts from 94 couples undergoing preimplantation genetic testing for aneuploidies (PGT-A), using fresh oocytes together with previously vitrified oocytes. Trophectoderm biopsies were performed and subjected to next generation sequencing. RESULTS: On average, 1.8 ± 1.0 oocyte vitrification cycles were performed per patient. Between fresh and vitrified cycles, no difference was observed between the number of fertilized oocytes (5.3 ± 4.2 versus 5.5 ± 3.0). Blastulation rate on day 5 per fertilized oocyte was significantly higher in the fresh group (62% ± 29% versus 44% ± 31%; p < 0.001). For the 504 biopsied blastocysts, 294 fresh versus 210 vitrified, no significant differences were found in the euploid rate, 40.5% versus 38.6% (p = 0.667), and mtDNA content, 30.1 (± 10.6) versus 30.0 (± 12.5) (p = 0.871), respectively. Regardless of the origin of the oocytes, aneuploid blastocysts contained significantly higher mtDNA values compared with the euploid ones (31.4 versus 28.0; p = 0.001). Furthermore, top-quality blastocysts had a significantly lower mtDNA content compared with moderate and poor-quality blastocysts (p < 0.001) and blastocysts biopsied on day 5 showed significantly lower mtDNA content compared with day 6 or day 7 blastocysts (p < 0.001). However, when analyzing the blastocyst mtDNA content according to the ploidy state, no differences were found for blastocyst quality or day of biopsy between blastocysts originating from fresh or vitrified oocytes. CONCLUSION: Oocyte vitrification does not affect the mtDNA content of trophectoderm biopsies.