Rescue in vitro maturation using ovarian support cells of human oocytes from conventional stimulation cycles yields oocytes with improved nuclear maturation and transcriptomic resemblance to in vivo matured oocytes.

PURPOSE: Determine if the gene expression profiles of ovarian support cells (OSCs) and cumulus-free oocytes are bidirectionally influenced by co-culture during in vitro maturation (IVM). METHODS: Fertility patients aged 25 to 45 years old undergoing conventional ovarian stimulation donated denuded immature oocytes for research. Oocytes were randomly allocated to either OSC-IVM culture (intervention) or Media-IVM culture (control) for 24-28 h. The OSC-IVM culture condition was composed of 100,000 OSCs in suspension culture with human chorionic gonadotropin (hCG), recombinant follicle stimulating hormone (rFSH), androstenedione, and doxycycline supplementation. The Media-IVM control lacked OSCs and contained the same supplementation. A limited set of in vivo matured MII oocytes were donated for comparative evaluation. Endpoints consisted of MII formation rate, morphological and spindle quality assessment, and gene expression analysis compared to in vitro and in vivo controls. RESULTS: OSC-IVM resulted in a statistically significant improvement in MII formation rate compared to the Media-IVM control, with no apparent effect on morphology or spindle assembly. OSC-IVM MII oocytes displayed a closer transcriptomic maturity signature to IVF-MII controls than Media-IVM control MII oocytes. The gene expression profile of OSCs was modulated in the presence of oocytes, displaying culture- and time-dependent differential gene expression during IVM. CONCLUSION: The OSC-IVM platform is a novel tool for rescue maturation of human oocytes, yielding oocytes with improved nuclear maturation and a closer transcriptomic resemblance to in vivo matured oocytes, indicating a potential enhancement in oocyte cytoplasmic maturation. These improvements on oocyte quality after OSC-IVM are possibly occurring through bidirectional crosstalk of cumulus-free oocytes and ovarian support cells.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

STUDY QUESTION: Can in vitro maturation (IVM) and developmental competence of human oocytes be improved by co-culture with ovarian support cells (OSCs) derived from human-induced pluripotent stem cells (hiPSCs)? SUMMARY ANSWER: OSC-IVM significantly improves the rates of metaphase II (MII) formation and euploid Day 5 or 6 blastocyst formation, when compared to a commercially available IVM system. WHAT IS KNOWN ALREADY: IVM has historically shown highly variable performance in maturing oocytes and generating oocytes with strong developmental capacity, while limited studies have shown a positive benefit of primary granulosa cell co-culture for IVM. We recently reported the development of OSCs generated from hiPSCs that recapitulate dynamic ovarian function in vitro. STUDY DESIGN, SIZE, DURATION: The study was designed as a basic science study, using randomized sibling oocyte specimen allocation. Using pilot study data, a prospective sample size of 20 donors or at least 65 oocytes per condition were used for subsequent experiments. A total of 67 oocyte donors were recruited to undergo abbreviated gonadotropin stimulation with or without hCG triggers and retrieved cumulus-oocyte complexes (COCs) were allocated between the OSC-IVM or control conditions (fetal-like OSC (FOSC)-IVM or media-only IVM) in three independent experimental design formats. The total study duration was 1 April 2022 to 1 July 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte donors between the ages of 19 and 37 years were recruited for retrieval after informed consent, with assessment of anti-Mullerian hormone, antral follicle count, age, BMI and ovarian pathology used for inclusion and exclusion criteria. In experiment 1, 27 oocyte donors were recruited, in experiment 2, 23 oocyte donors were recruited, and in experiment 3, 17 oocyte donors and 3 sperm donors were recruited. The OSC-IVM culture condition was composed of 100 000 OSCs in suspension culture with hCG, recombinant FSH, androstenedione, and doxycycline supplementation. IVM controls lacked OSCs and contained either the same supplementation, FSH and hCG only (a commercial IVM control), or FOSCs with the same supplementation (Media control). Experiment 1 compared OSC-IVM, FOSC-IVM, and a Media control, while experiments 2 and 3 compared OSC-IVM and a commercial IVM control. Primary endpoints in the first two experiments were the MII formation (i.e. maturation) rate and morphological quality assessment. In the third experiment, the fertilization and embryo formation rates were assessed with genetic testing for aneuploidy and epigenetic quality in blastocysts. MAIN RESULTS AND THE ROLE OF CHANCE: We observed a statistically significant improvement (∼1.5×) in maturation outcomes for oocytes that underwent IVM with OSCs compared to control Media-IVM and FOSC-IVM in experiment 1. More specifically, the OSC-IVM group yielded a MII formation rate of 68% ± 6.83% SEM versus 46% ± 8.51% SEM in the Media control (P = 0.02592, unpaired t-test). FOSC-IVM yielded a 51% ± 9.23% SEM MII formation rate which did not significantly differ from the media control (P = 0.77 unpaired t-test). Additionally, OSC-IVM yielded a statistically significant ∼1.6× higher average MII formation rate at 68% ± 6.74% when compared to 43% ± 7.90% in the commercially available IVM control condition (P = 0.0349, paired t-test) in experiment 2. Oocyte morphological quality between OSC-IVM and the controls did not significantly differ. In experiment 3, OSC-IVM oocytes demonstrated a statistically significant improvement in Day 5 or 6 euploid blastocyst formation per COC compared to the commercial IVM control (25% ± 7.47% vs 11% ± 3.82%, P = 0.0349 logistic regression). Also in experiment 3, the OSC-treated oocytes generated blastocysts with similar global and germline differentially methylated region epigenetic profiles compared commercial IVM controls or blastocysts after either conventional ovarian stimulation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: While the findings of this study are compelling, the cohort size remains limited and was powered on preliminary pilot studies, and the basic research nature of the study limits generalizability compared to randomized control trials. Additionally, use of hCG-triggered cycles results in a heterogenous oocyte cohort, and potential differences in the underlying maturation state of oocytes pre-IVM may limit or bias findings. Further research is needed to clarify and characterize the precise mechanism of action of the OSC-IVM system. Further research is also needed to establish whether these embryos are capable of implantation and further development, a key indication of their clinical utility. WIDER IMPLICATIONS OF THE FINDINGS: Together, these findings demonstrate a novel approach to IVM with broad applicability to modern ART practice. The controls used in this study are in line with and have produced similar to findings to those in the literature, and the outcome of this study supports findings from previous co-culture studies that found benefits of primary granulosa cells on IVM outcomes. The OSC-IVM system shows promise as a highly flexible IVM approach that can complement a broad range of stimulation styles and patient populations. Particularly for patients who cannot or prefer not to undergo conventional gonadotropin stimulation, OSC-IVM may present a viable path for obtaining developmentally competent, mature oocytes. STUDY FUNDING/COMPETING INTEREST(S): A.D.N., A.B.F., A.G., B.P., C.A., C.C.K., F.B., G.R., K.S.P., K.W., M.M., P.C., S.P., and M.-J.F.-G. are shareholders in the for-profit biotechnology company Gameto Inc. P.R.J.F. declares paid consultancy for Gameto Inc. P.C. also declares paid consultancy for the Scientific Advisory Board for Gameto Inc. D.H.M. has received consulting services from Granata Bio, Sanford Fertility and Reproductive Medicine, Gameto, and Buffalo IVF, and travel support from the Upper Egypt Assisted Reproduction Society. C.C.K., S.P., M.M., A.G., B.P., K.S.P., G.R., and A.D.N. are listed on a patent covering the use of OSCs for IVM: U.S. Provisional Patent Application No. 63/492,210. Additionally, C.C.K. and K.W. are listed on three patents covering the use of OSCs for IVM: U.S. Patent Application No. 17/846,725, U.S Patent Application No. 17/846,845, and International Patent Application No.: PCT/US2023/026012. C.C.K., M.P.S., and P.C. additionally are listed on three patents for the transcription factor-directed production of granulosa-like cells from stem cells: International Patent Application No.: PCT/US2023/065140, U.S. Provisional Application No. 63/326,640, and U.S. Provisional Application No. 63/444,108. The remaining authors have no conflicts of interest to declare.

Impact of whole systems traditional Chinese medicine on in-vitro fertilization outcomes.

Patients undergoing IVF may receive either acupuncture or whole-systems traditional Chinese medicine (WS-TCM) as an adjuvant IVF treatment. WS-TCM is a complex intervention that can include acupuncture, Chinese herbal medicine, dietary, lifestyle recommendations. In this retrospective cohort study, 1231 IVF patient records were reviewed to assess the effect of adjuvant WS-TCM on IVF outcomes compared among three groups: IVF with no additional treatment; IVF and elective acupuncture on day of embryo transfer; or IVF and elective WS-TCM. The primary outcome was live birth. Of 1069 non-donor cycles, WS-TCM was associated with greater odds of live birth compared with IVF alone (adjusted odds ratio [AOR] 2.09; 95% confidence interval [CI] 1.36 to 3.21), or embryo transfer with acupuncture only (AOR 1.62; 95% CI 1.04 to 2.52). Of 162 donor cycles, WS-TCM was associated with increased live births compared with all groups (odds Ratio [OR] 3.72; 95% CI 1.05 to 13.24, unadjusted) or embryo transfer with acupuncture only (OR 4.09; 95% CI: 1.02 to 16.38, unadjusted). Overall, IVF with adjuvant WS-TCM was associated with greater odds of live birth in donor and non-donor cycles. These results should be taken cautiously as more rigorous research is needed.

Resultados del uso de un sistema cerrado (S3) en la vitrificación de blastocistos humanos / Results following the use of a closed system (S3) for vitrification of human blastocysts

Este artículo describe el uso de un sistema cerrado de vitrificación que es fácil de usar y permite que los blastocitos sean vitrificados en un recipiente seguro y cerrado. Este sistema no utiliza dimetil sulfóxido (DMSO) como crío-protector y se le denomina vitrificación S3. Blastocistos humanos fueron vitrificados en los días 5 y 6 de desarrollo (día de recuperación es igual a día 0). Se conservó solo blastocistos completos de alta calidad o avanzados en el desarrollo. Se realizó un total de 139 transferencias, con una tasa de supervivencia global de 81%. La tasa de embarazos nacidos y en curso es de 58%, con una tasa de implantación de 48% para todos los pacientes en todos los grupos de edad. Los resultados globales obtenidos con este sistemacerrado son alentadores.

This paper describes the use of a closed vitrification system that is easy to use and allows the blastocysts to be vitrified in a closed secure container. This system does not use dimethylsulphoxide (DMSO) as a cryoprotectant as well. This system is referred to as S3 vitrification. Human blastocysts were vitrified on Days 5 and 6 (Day ofretrieval equals Day 0). Only high quality full blastocysts or greater in development were preserved. A total of 139 transfers have been performed with an overall survival rate of 81%. The delivery/ongoing pregnancy rates are 58% with a 48% implantation rate for all patients in all age groups. The overall outcomes achieved with this closed system are encouraging.

A new safe, simple and successful vitrification method for bovine and human blastocysts.

This study examined a new method for vitrification of blastocysts that is safe, simple and easy to learn and use. Current vitrification techniques have shortcomings that include the use of dimethyl sulphoxide, one of the more toxic cryoprotectants, and minute containers that are difficult to handle and are usually open to contamination. Cell handling and loading times are very short, which allows no room for user-associated errors and increases the difficulty of the procedure. This study describes a method of vitrification without these shortcomings. Human and bovine blastocysts were exposed to a series of three cryoprotectant solutions and loaded into a 0.25 ml sterile straw, heat sealed at both ends and vitrified. This technique allowed sufficient time for cryoprotectant exposure, loading, sealing and vitrification. Research blastocysts were thawed, cultured for 24 h, and stained for cell viability. The majority survived and on average had few lysed cells. In clinical studies from three different centres, 81.4% of vitrified blastocysts were intact after thawing. Out of 43 transfers with 76 blastocysts replaced, 44.7% implanted, 43.4% yielded a fetal heart beat, and a total of 32 babies have been delivered or are ongoing. The overall clinical pregnancy per transfer rate was 60.4%. The high survival rates and clinical pregnancy rates obtained with this new, safe and easy-to-use vitrification procedure are encouraging.

The limited importance of pronuclear scoring of human zygotes.

BACKGROUND: Several studies have shown a correlation between the pronuclear morphology score (PNMS) and subsequent embryo development and implantation. Embryos with poor pronuclear score, elsewhere referred to as Z3 and Z4, are often not transferred or cryopreserved because it is believed that they have poor pregnancy potential. The objective of this study is to report our data on the use of the pronuclear score and its effect on pregnancy outcome. METHODS: Retrospective analysis of IVF/ICSI-embryo transfer cycles completed over the course of 1 year (n = 334). Comparisons were made only in those groups of patients in whom cohorts of similarly scored PNMS embryos were transferred. The proportion of such homologous cohorts was 104/334 (31%). All other replacements were excluded from final analysis as they were dissimilar as far as PNMS is concerned. Pregnancy outcomes were evaluated. RESULTS: The incidence of live birth resulting from the transfer of single pronuclear score homologous embryo types was 56 (14/25), 41 (13/32), 54 (23/43) and 0% (0/4) for PNMS scores 1, 2, 3 and 4, respectively. There was no correlation between PNMS category of the embryos transferred and live birth rates (P = 0.139). CONCLUSIONS: PNMSs of 1, 2 or 3 do not correlate with live birth rates when assessing unique PNMS embryo transfers. In particular, previously considered poor (type 3) embryos can result in pregnancy with normal live birth rates. Whether type 4 embryos are compatible with normal development remains to be shown.

Embryonic development and pregnancies following sequential culture in human tubal fluid and a modified simplex optimized medium containing amino acids.

Pregnancies following human blastocyst transfers were established using a protein supplemented modified potassium simplex optimized medium containing amino acids (KSOM(AA)). Zygotes were first cultured for 2 days in protein supplemented human tubal fluid medium. Resulting embryos (day 3) were subsequently cultured in protein supplemented KSOM(AA) until day 5 of development. Pregnancy and implantation rates were noted after culture and transfer of blastocysts following this culture scheme. The impact of conventional insemination, intracytoplasmic sperm injection (ICSI) and maternal age on developmental rates as well as pregnancy rates was also evaluated. Maternal age had a significant impact (P < 0.05) on developmental rates of embryos until day 3 of culture. Overall, rate of blastocyst development for cultured day 3 embryos was 62%. Patients >39 years of age had lower (P < 0.05) rates of blastocyst development than patients in the younger subgroups. ICSI had no impact on developmental rates until day 3 of culture or rate of blastocyst development. Ongoing pregnancy and implantation rates following culture in KSOM(AA) were 51 and 37% respectively. These results indicate that KSOM(AA) supports high rate blastocyst development and resulting pregnancy rates.

Fertilization, embryonic development and pregnancy losses with intracytoplasmic sperm injection for surgically-retrieved spermatozoa.

Prior to the development of intracytoplasmic sperm injection (ICSI), azoospermic and severely oligozoospermic men had little to no chance of having a biological child. In this study, ICSI outcome in 454 transfers with ejaculated spermatozoa and 59 transfers with surgically retrieved spermatozoa were evaluated. Normal fertilization rate using ejaculated spermatozoa was 75% of 5995 oocytes, and 73% of 751 oocytes for surgically retrieved spermatozoa; with ongoing pregnancy rates of 53% (242/454) and 61% (36/59) respectively. Surgically retrieved spermatozoa significantly (P < 0.05) impacted 1PN oocytes (6.1%, 46/751), severely fragmented embryos (8.8%, 46/550) and incidence of pregnancy loss (11%, 4/36). When using ejaculated spermatozoa, incidence of 1PN oocytes, severely fragmented embryos and pregnancy loss was 2.9% (177/5995), 4.5% (200/4365), 2.4% (6/242) respectively.