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Objective: To understand the clinical risks associated with the transfer of embryos classified as a mosaic using preimplantation genetic testing for aneuploidy., Design: Analysis of data collected between 2017 and 2023., Setting: Multicenter., Patients: Patients of infertility treatment., Intervention: Comparison of pregnancies resulting from embryos classified as euploid or mosaic using the 20%-80% interval in chromosomal intermediate copy numbers to define a mosaic result., Main Outcome Measures: Rates of spontaneous abortion, birth weight, length of gestation, incidence of birth defects, and chromosomal status during gestation., Results: Implanted euploid embryos had a significantly lower risk of spontaneous abortion compared with mosaic embryos (8.9% [n = 8,672; 95% confidence interval {CI95} 8.3, 9.5] vs. 22.2% [n = 914; CI95 19.6, 25.0]). Embryos with mosaicism affecting whole chromosomes (not segmental) had the highest risk of spontaneous abortion (27.6% [n = 395; CI95 23.2, 32.3]). Infants born from euploid, mosaic, and whole chromosome mosaic embryos had average birth weights and lengths of gestation that were not statistically different (3,118 g and 267 days [n = 488; CI95 3,067, 3,169, and 266, 268], 3052 g and 265 days [n = 488; CI95 2,993, 3,112, and 264,267], 3,159 g and 268 days [n = 194; CI95 3,070, 3,249, and 266,270], respectively). Out of 488 infants from mosaic embryo transfers (ETs), one had overt gross abnormalities as defined by the Centers for Disease Control and Prevention. Most prenatal tests performed on pregnancies from mosaic ETs had normal results, and only three pregnancies produced prenatal test results reflecting the mosaicism detected at the embryonic stage (3 out of 250, 1.2%; CI95 0.25, 3.5)., Conclusion: Although embryos classified as mosaic experience higher rates of miscarriage than euploid embryos (with a particularly high frequency shortly after implantation), infants born of mosaic ETs are similar to infants of euploid ETs. Prenatal testing indicates that mosaicism resolves during most pregnancies, although this process is not perfectly efficient. In a small percentage of cases, the mosaicism persists through gestation. These findings can serve as risk-benefit considerations for mosaic ETs in the fertility clinic., Competing Interests: Declaration of interests M.V. has nothing to disclose. E.G. has nothing to disclose. J.A.G. has nothing to disclose. M.M. has nothing to disclose. C.L. reports Patent application - Detection of structural aberrations in embryos, Patent application -Method for non-invasive preimplantation genetic diagnosis. M.C. has nothing to disclose. S.K. has nothing to disclose. P.Y. has nothing to disclose. N.K. has nothing to disclose. L.C. has nothing to disclose. A.B. has nothing to disclose. E.H.C. has nothing to disclose. C.Y.S. has nothing to disclose. M.S.L. has nothing to disclose. M.D.B. has nothing to disclose. A.R.C. reports honoraria from CooperSurgical and Ferring; leadership or board position Midwest Reproductive Society International, Sunfish, and Celmatix; stock Kindbody, Sunfish, Celmatix. Author Besser report honoraria from American Society for Reproductive Medicine, American College for Medical Genetics, Canadian Fertility and Andrology Society, Illinois Society of Genetic Professionals, Collaborative Group of the Americas on Inherited Colorectal Cancer, and National Society of Genetic Counselors; travel support from Collaborative Group of the Americas on Inherited Colorectal Cancer and American College for Medical Genetics; board member Genetic Counseling Professional Group (ASRM), Patient Education Committee (ASRM), International Registry of Mosaic Embryo Transfers. D.K.G. reports funding from Cooper Surgical and Igenomix for the submitted work; funding from Cooper Surgical; consulting fees from Care Fertility; honoraria from Ferring; payment for expert testimony; travel support from Ferring; Chair of International Chromosome and genome society; stock options from Conceivable outside the submitted work. D.Y.T. has nothing to disclose. F.L.B. has nothing to disclose. C.G.Z. has nothing to disclose. A.R.V. has nothing to disclose. A.G.B. has nothing to disclose. S.M. reports Patent application - Detection of structural aberrations in embryos, Patent application -Method for non-invasive preimplantation genetic diagnosis; Board Director – International Society for Preimplantation Diagnosis (PGDIS); Board Director – Canadian Fertility and Andrology Society (CFAS). F.S. has nothing to disclose., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The spread of SARS-CoV-2 has led to a devastating pandemic, with infections resulting in a range of symptoms collectively known as COVID-19. The full repertoire of human tissues and organs susceptible to infection is an area of active investigation, and some studies have implicated the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood, and particularly the impact on early embryogenesis and establishment of a pregnancy are not known. In this work, we explore the susceptibility of early human embryos to SARS-CoV-2 infection. By using RNA-seq and immunofluorescence, we note that ACE2 and TMPRSS2, two canonical cell entry factors for SARS-CoV-2, are co-expressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos. For the purpose of viral entry studies, we used fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, and we observe robust infection of trophectoderm cells. This permissiveness could be attenuated with blocking antibodies targeting S or ACE2. When exposing human blastocysts to the live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath., (© 2022. The Author(s).)

Extra or missing chromosomes-a phenomenon termed aneuploidy-frequently arise during human meiosis and embryonic mitosis and are the leading cause of pregnancy loss, including in the context of in vitro fertilization (IVF). While meiotic aneuploidies affect all cells and are deleterious, mitotic errors generate mosaicism, which may be compatible with healthy live birth. Large-scale abnormalities such as triploidy and haploidy also contribute to adverse pregnancy outcomes, but remain hidden from standard sequencing-based approaches to preimplantation genetic testing for aneuploidy (PGT-A). The ability to reliably distinguish meiotic and mitotic aneuploidies, as well as abnormalities in genome-wide ploidy, may thus prove valuable for enhancing IVF outcomes. Here, we describe a statistical method for distinguishing these forms of aneuploidy based on analysis of low-coverage whole-genome sequencing data, which is the current standard in the field. Our approach overcomes the sparse nature of the data by leveraging allele frequencies and linkage disequilibrium (LD) measured in a population reference panel. The method, which we term LD-informed PGT-A (LD-PGTA), retains high accuracy down to coverage as low as 0.05 × and at higher coverage can also distinguish between meiosis I and meiosis II errors based on signatures spanning the centromeres. LD-PGTA provides fundamental insight into the origins of human chromosome abnormalities, as well as a practical tool with the potential to improve genetic testing during IVF.

Chromosomal mosaicism, the coexistence of cells with different chromosomal content, has been documented in human embryos for 3 decades. Early versions of preimplantation genetic testing for aneuploidy (PGT-A) did not measure mosaicism, either because typically only a single cell was assessed or because the technique could not accurately identify it. Although this led to a straightforward diagnosis (an embryo was considered either normal or abnormal), it simply avoided the issue and, in hindsight, may have led to numerous misdiagnoses with negative clinical consequences. Modern PGT-A evaluates a multicellular biopsy specimen with techniques capable of recognizing intermediate copy number signals for chromosomes or subchromosomal regions. We are, therefore, inevitably confronted with the issue of mosaicism and the challenge of managing embryos producing such results in the clinic. Here we discuss recent data showing that not only mosaicism in general, but specific features of mosaicism detected with PGT-A, are associated with variable clinical outcomes. The conclusion is evident: mosaicism should be considered for more informed and improved embryo selection in the clinic., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To study how the attributes of mosaicism identified during preimplantation genetic testing for aneuploidy relate to clinical outcomes, in order to formulate a ranking system of mosaic embryos for intrauterine transfer., Design: Compiled analysis., Setting: Multi-center., Patient(s): A total of 5,561 euploid blastocysts and 1,000 mosaic blastocysts used in clinical transfers in patients undergoing fertility treatment., Intervention(s): None., Main Outcome Measure(s): Implantation (gestational sac), ongoing pregnancy, birth, and spontaneous abortion (miscarriage before 20 weeks of gestation)., Result(s): The euploid group had significantly more favorable rates of implantation and ongoing pregnancy/birth (OP/B) compared with the combined mosaic group or the mosaic group affecting only whole chromosomes (implantation: 57.2% vs. 46.5% vs. 41.8%; OP/B: 52.3% vs. 37.0% vs. 31.3%), as well as lower likelihood of spontaneous abortion (8.6% vs. 20.4% vs. 25%). Whole-chromosome mosaic embryos with level (percent aneuploid cells) <50% had significantly more favorable outcomes than the ≥50% group (implantation: 44.5% vs. 30.4%; OP/B: 36.1% vs. 19.3%). Mosaic type (nature of the aneuploidy implicated in mosaicism) affected outcomes, with a significant correlation between number of affected chromosomes and unfavorable outcomes. This ranged from mosaicism involving segmental abnormalities to complex aneuploidies affecting three or more chromosomes (implantation: 51.6% vs. 30.4%; OP/B: 43.1% vs. 20.8%). Combining mosaic level, type, and embryo morphology revealed the order of subcategories regarding likelihood of positive outcome., Conclusion(s): This compiled analysis revealed traits of mosaicism identified with preimplantation genetic testing for aneuploidy that affected outcomes in a statistically significant manner, enabling the formulation of an evidence-based prioritization scheme for mosaic embryos in the clinic., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Germline genome editing (GGE) holds the potential to mitigate or even eliminate human heritable genetic disease, but also carries genuine risks if not appropriately regulated and performed. It also raises fears in some quarters of apocalyptic scenarios of designer babies that could radically change human reproduction. Clinical need and the availability of alternatives are key considerations in the ensuing ethical debate. Writing from the perspective of a fertility clinic, we offer a realistic projection of the demand for GGE. We lay out a framework proposing that GGE, hereditary genetic disorders, and in vitro fertilization are fundamentally entwined concepts. We note that the need for GGE to cure heritable genetic disease is typically grossly overestimated, mainly due to the underappreciated role of preimplantation genetic testing. However, we might still find applications for GGE in the correction of chromosomal abnormalities in early embryos, but techniques for that purpose do not yet exist.

Objective: To investigate the parameters of mosaicism and the biological mechanisms leading to healthy pregnancies from mosaic embryo transfers., Design: Prospective study., Setting: IVF center and associated research laboratory., Patient(s): Fifty-nine patients., Intervention(s): Embryos underwent blastocyst-stage preimplantation genetic testing for aneuploidy by next-generation sequencing. Trophectoderm biopsies containing 20%-80% abnormal cells were deemed mosaic, and corresponding blastocysts were transferred. Mosaic embryos donated to research were examined for karyotype concordance in multiple biopsies and assessed for cell proliferation and death by immunofluorescence and computational quantitation., Main Outcome Measure(s): Chemical start of pregnancy, implantation, fetal heartbeat, and birth., Result(s): Globally, mosaic embryos showed inferior clinical outcomes compared with euploid embryos. Aneuploid cell percentage in trophectoderm biopsies did not correlate with outcomes, but type of mosaicism did, as embryos with single mosaic segmental aneuploidies fared better than all other types. Mosaic blastocysts generated from oocytes retrieved at young maternal ages (≤34 years) showed better outcomes than those retrieved at older maternal ages. Mosaic embryos displayed low rates of karyotype concordance between multiple biopsies and showed significant elevation of cell proliferation and death compared with euploid embryos., Conclusion(s): After euploid embryos, mosaic embryos can be considered for transfer, prioritizing those of the single segmental mosaic type. If a patient has mosaic embryos available that were generated at different ages, preference should be given to those made at younger ages. Intrablastocyst karyotype discordance and differential cell proliferation and death might be reasons that embryos classified as mosaic can result in healthy pregnancies and babies., (Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Study Question: Is a clinical trophectoderm (TE) biopsy a suitable predictor of chromosomal aneuploidy in blastocysts?, Summary Answer: In the analyzed group of blastocysts, a clinical TE biopsy was an excellent representative of blastocyst karyotype in cases of whole chromosome aneuploidy, but in cases of only segmental (sub-chromosomal) aneuploidy, a TE biopsy was a poor representative of blastocyst karyotype., What Is Known Already: Due to the phenomenon of chromosomal mosaicism, concern has been expressed about the possibility of discarding blastocysts classified as aneuploid by preimplantation genetic testing for aneuploidy (PGT-A) that in fact contain a euploid inner cell mass (ICM). Previously published studies investigating karyotype concordance between TE and ICM have examined small sample sizes and/or have utilized chromosomal analysis technologies superseded by Next Generation Sequencing (NGS). It is also known that blastocysts classified as mosaic by PGT-A can result in healthy births. TE re-biopsy of embryos classified as aneuploid can potentially uncover new instances of mosaicism, but the frequency of such blastocysts is currently unknown., Study Design, Size, Duration: For this study, 45 patients donated 100 blastocysts classified as uniform aneuploids (non-mosaic) using PGT-A by NGS (n = 93 whole chromosome aneuploids, n = 7 segmental aneuploids). In addition to the original clinical TE biopsy used for PGT-A, each blastocyst was subjected to an ICM biopsy as well as a second TE biopsy. All biopsies were processed for chromosomal analysis by NGS, and karyotypes were compared to the original TE biopsy., Participants/materials, Setting, Methods: The setting for this study was a single IVF center with an in-house PGT-A program and associated research laboratory., Main Results and the Role of Chance: When one or more whole chromosomes were aneuploid in the clinical TE biopsy, the corresponding ICM was aneuploid in 90 out of 93 blastocysts (96.8%). When the clinical TE biopsy contained only segmental (sub-chromosomal) aneuploidies, the ICM was aneuploid in three out of seven cases (42.9%). Blastocysts showing aneuploidy concordance between clinical TE biopsy and ICM were also aneuploid in a second TE biopsy in 86 out of 88 cases (97.7%). In blastocysts displaying clinical TE-ICM discordance, a second TE biopsy was aneuploid in only two out of six cases (33.3%)., Limitations, Reasons for Caution: All embryos in this study had an initial classification of 'aneuploid' and not 'euploid' or 'mosaic'. Therefore, the findings of this study refer specifically to a TE biopsy predicting aneuploidy in the remaining blastocyst, and cannot be extrapolated to deduce the ability of a TE biopsy to predict euploidy in the blastocyst. No conclusions should be drawn from this study about the ability of a mosaic TE biopsy to predict the karyotype of the corresponding blastocyst. Caution should be exercised in generalizing the findings of the sample group of this study to the general IVF blastocyst population. The segmental aneuploidy group only contained seven samples., Wider Implications of the Findings: The high rate of intra-blastocyst concordance observed in this study concerning whole chromosome aneuploidy contributes experimental evidence to the validation of PGT-A at the blastocyst stage. Concomitantly, the results suggest potential clinical value in reassessing blastocysts deemed aneuploid by TE re-biopsy in select cases, particularly in instances of segmental aneuploidies. This could impact infertility treatment for patients who only have blastocysts classified as aneuploid by PGT-A available., Study Funding/competing Interest(s): This study was supported by the Zouves Foundation for Reproductive Medicine and Zouves Fertility Center. The authors have no competing interest to disclose., Trial Registration Number: Not applicable.

Behind every successful IVF embryo transfer, there is a great game of chance. Methods seeking to tilt the balance and increase the likelihood of implantation have been proposed and implemented with varying results, including embryo morphology, FISH-PGS, comprehensive chromosomal screening (CCS), morphokinetics, endometrial receptivity testing. It has been suggested that mitochondrial DNA (mtDNA) copy number could serve as a biomarker for embryo viability, but this concept was recently challenged. The world of IVF is left with unanswered questions: Why are there discrepancies in the reports? Should mtDNA copy number be considered to rank embryos for transfer? And in a broader sense, how well must a technique be validated before its implementation in the IVF clinic? Here, we explore these questions attempting to piece together the published data and suggest future directions to help unravel the subject matter.

Objective: To accurately determine mitochondrial DNA (mtDNA) levels in human blastocysts., Design: Retrospective analysis., Setting: IVF clinic., Patient(s): A total of 1,396 embryos derived from 259 patients., Intervention(s): Blastocyst-derived trophectoderm biopsies were tested by next-generation sequencing (NGS) and quantitative real-time polymerase chain reaction (qPCR)., Main Outcome Measure(s): For each sample the mtDNA value was divided by the nuclear DNA value, and the result was further subjected to mathematical analysis tailored to the genetic makeup of the source embryo., Result(s): On average the mathematical correction factor changed the conventionally determined mtDNA score of a given blastocyst via NGS by 1.43% ± 1.59% (n = 1,396), with maximal adjustments of 17.42%, and via qPCR by 1.33% ± 8.08% (n = 150), with maximal adjustments of 50.00%. Levels of mtDNA in euploid and aneuploid embryos showed a statistically insignificant difference by NGS (euploids n = 775, aneuploids n = 621) and by qPCR (euploids n = 100, aneuploids n = 50). Blastocysts derived from younger or older patients had comparable mtDNA levels by NGS ("young" age group n = 874, "advanced" age group n = 514) and by qPCR ("young" age group n = 92, "advanced" age group n = 58). Viable blastocysts did not contain significantly different mtDNA levels compared with unviable blastocysts when analyzed by NGS (implanted n = 101, nonimplanted n = 140) and by qPCR (implanted n = 49, nonimplanted n = 51)., Conclusion(s): We recommend implementation of the correction factor calculation to laboratories evaluating mtDNA levels in embryos by NGS or qPCR. When applied to our in-house data, the calculation reveals that overall levels of mtDNA are largely equal between blastocysts stratified by ploidy, age, or implantation potential., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the efficacy of zygote intrafallopian transfer (ZIFT) versus standard IVF-ET for the treatment of nontubal factor infertility., Design: A prospective randomized trial., Participants: Fifty-nine couples undergoing oocyte retrieval for nontubal infertility in a university hospital-based IVF-ET program., Interventions: A maximum of four cleaving embryos were transferred into the fallopian tube or uterine cavity 48 or 55 hours after oocyte retrieval, respectively., Main Outcome Measures: Rates of implantation, pregnancy, and spontaneous abortion (SAB) were compared., Results: Clinical pregnancies occurred after 26.5% versus 12% of retrievals and 29% versus 14.3% of transfers in IVF-ET versus ZIFT cycles, respectively. Pregnancy, implantation, and SAB rates did not differ between the groups., Conclusions: This prospective randomized trial failed to demonstrate any therapeutic improvement associated with the increased complexity of ZIFT as compared with standard IVF-ET.

Objectives: To determine the extent of contamination of ovarian follicular fluid and serum samples in women undergoing in-vitro fertilization and to study the effect of the contaminants on reproductive outcome., Design: Inception cohort study., Patients: Seventy-four women undergoing in-vitro fertilization at three regional clinics in Halifax, Hamilton, Ont., and Vancouver., Main Outcome Measures: Follicular fluid and serum levels of contaminants, cleavage rates and time to cleavage of first egg., Results: Five chlorinated organic chemicals were frequently found in the two types of samples: alpha-chlordane (ALCH), dichlorochlorophenylethylene (DDE), heptachloroepoxide-oxychlordane (OXCH), hexachlorobenzene (HCB) and polychlorinated biphenyl (PCB). The levels were generally low. Regional differences between the three clinics were present. Samples from the Halifax clinic had the lowest frequency and level of contamination. The source of drinking water (well, bottled or municipal) was an important confounder. The concentrations of the five contaminants did not affect the cleavage rate or the time to cleavage of the first egg., Conclusion: Trace amounts of toxic and persistent chlorinated organic chemicals found in the follicular fluid of Canadian women undergoing in-vitro fertilization did not seem to have any adverse biologic effect on the rate of fertilization and the time to cleavage. Reasons for regional differences in the concentrations of contaminants require further study.

Objective: To assess the outcome of in vitro fertilization and embryo transfer (IVF-ET) in women with refractory polycystic ovarian syndrome (PCOS)., Design: Retrospective case series with an age-matched control group., Setting: Ovulation induction and IVF programs in a tertiary referral center., Patients and Interventions: Nine patients with PCOS who failed standard ovulation induction treatment (clomiphene citrate plus greater than or equal to 6 ovulatory human menopausal gonadotropin [hMG] cycles) underwent 19 cycles of IVF-ET. Forty age-matched tubal factor patients who completed 40 cycles of IVF-ET served as a control group., Outcome Measures: Demographic features and IVF-ET cycle characteristics were compared using Student's t-test and Fisher's exact test., Results: Cycles of IVF-ET in patients with PCOS were associated with higher estradiol levels (5,222 versus 4,009 pmol/L), lower hMG requirements (15.8 versus 19.6 vials), greater numbers of oocytes (7.6 versus 5.6), and lower fertilization rates (56% versus 75%) compared with tubal factor cycles (P less than 0.05). However, the number of embryos transferred (3.9 versus 4.0) and the clinical pregnancy rate per embryo transfer (24% versus 25%) did not differ significantly between the two groups., Conclusion: These results suggest that conception failure after six or more ovulatory hMG cycles in patients with PCOS does not adversely affect subsequent IVF performance.

Data were collected from self-administered questionnaires returned by 33 female participants and 18 of their partners in the University of British Columbia's in Vitro Fertilization/Gamete Intrafallopian Transfer (IVF/GIFT) Program during a 2-month period. Emotional reactions to each of the stages of IVF/GIFT by treatment phase were measured. Responses were then grouped into the following categories: anxiety, depression, loss of control, and positive feelings. For female participants, anxiety was reported most frequently throughout the treatment process and loss of control was highest following embryo replacement. Male and female participants reported high rates of depression at the completion of the treatment cycle. The findings from this pilot study outline the emotional experiences of male and female participants undergoing IVF/GIFT by treatment phase and indicate their desire for support services.

Patients with recurrent spontaneous abortions have been successfully treated in many centers with third-party immunization directed to a putative TLX antigen system. This immunotherapy requires the screening of a large number of donors to match the patients' red blood cell (RBC) phenotype and has the potential risks associated with transfusions from 30 to 50 donors. Our modified approach to third-party immunization is to use irradiated frozen-stored purified lymphocytes pooled from five normal donors. Mononuclear cells from normal donors are obtained in a cell separator. After sedimentation and Ficoll-Hypaque separation, the cells are stored in liquid N2. The RBC depletion of the final preparation is of the order of 5 to 6 logs, theoretically decreasing the need for RBC phenotyping except for the Rh system. Using a highly sensitive fluorescence-activated cell sorter technique and an ADCC assay, we found that ABH, Rh, Fya Fyb, Jka Jkb, MNS, and Kell antigens are either not expressed by cryopreserved human mononuclear cells, or, if so, they are below the level of detection of these highly sensitive assays. We conclude that the use of pooled frozen mononuclear cells is an adequate alternative for immunotherapy. It decreases the transfusion risks associated with exposure to a large number of donors and the need for RBC phenotyping, making this modality of treatment more accessible.