Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos.

Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targeted GENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.

Composition and distribution of fatty acids in various lipid fractions in serum and follicular fluid of women undergoing assisted reproductive technology.

Fatty acids (FA) in follicular fluid (FF) are present in an esterified form [triglycerides, cholesterol esters and phospholipids] or as non-esterified FA, which partly originate from blood. However, a comprehensive comparison of blood vs. FF FA in various lipid classes is missing. The aim of this study was to determine the distribution of the FA composition in each lipid class of serum and FF, and to investigate their mutual correlations. A total of 74 patients undergoing assisted reproductive technology treatment were involved in the study. Both in serum as well as FF, saturated FA and mono-unsaturated FA were predominant in non-esterified FA and triglycerides fractions while poly-unsaturated FA were mainly present in phospholipids and cholesterol esters fractions, although phospholipids also contained high proportions of saturated FA. Irrespective of the lipid class, the FA proportions differed between serum and FF (P < 0.05). Despite these differences, most of the FA in triglycerides, phospholipids and cholesterol esters of FF were well correlated with their proportions in serum. Nevertheless, only weak to moderate associations (r < 0.60) were observed for the majority of the FA in the non-esterified FA fraction. Differences in FA product/precursor-ratios were found between serum and FF, such as higher C20:4n-6 to C18:2n-6 and C20:5n-3 to C18:3n-3 in FF. FA metabolism (e.g. desaturation and elongation) takes place in cells of the intrafollicular micro-environment. Moreover, good correlations between esterified FA in serum and FF suggest esterified FA in blood could be representative of esterified FA in FF.

The fatty acid composition in follicles is related to the developmental potential of oocytes up to the blastocyst stage: a single-centre cohort study.

BACKGROUND: Advanced maternal age and obesity are associated with impaired female fertility. Moreover, fatty acids (FA) in follicular fluid (FF) play important roles in oocyte maturation and embryo development. However, the effects of body mass index (BMI), age, and FF FA composition on embryo development between days 3 and 5 and blastocyst stage on day 5 are still unclear. METHODS: This study included 138 patients undergoing assisted reproductive technology (ART), which were divided into three BMI groups (18.5-24.9 kg/m2 vs. 25.0-29.9 kg/m2 vs. ≥ 30.0 kg/m2) and three age-related groups (20-30 years vs. 31-34 years vs. ≥ 35 years) which were compared for ART outcomes. Further, observations were divided into quartiles based on either of three parameters related to embryo outcome, i.e. (i) embryos developing between days 3 and 5 (ED3-5) and (ii) expanded blastocysts on day 5 (EB5), both expressed proportionally to the number of oocytes with two pronuclei (2PN), as well as (iii) the embryo utilization rate (EUR). Proportions of FF FA were then compared between Q1 and Q4, representing the quartile with the worst vs. the best embryo outcome, respectively. Finally, regression models were created to assess the relationships between BMI, age, FF total FA (TFA) concentration, relative proportions of specific FA and embryo outcome. RESULTS: Patients of Q1 had higher proportions of FF C20:5n-3, C22:6n-3 and total n-3 PUFA than Q4 patients. Furthermore, Q4 patients tended to be younger than Q1 patients. Within the whole cohort, the proportion of C20:5n-3 negatively correlated with ED3-5/2PN and EUR, while EB5/2PN tended to be negatively correlated with age. Regression models within the overweight and obese group confirmed the negative relation between C20:5n-3 and ED3-5/2PN, but also indicated additional associations: C18:1n-9 and C20:4n-6 were positively associated with ED3-5/2PN and EUR, respectively while the proportion of C18:0 was negatively associated with EUR. CONCLUSION: The proportions of n-3 PUFA, particularly C20:5n-3 and C22:6n-3 were reduced in the patients' quartile with the best embryo outcome. This group of patients was also younger. However, the embryo quality parameters of overweight/obese patients were not associated with age but were positively associated with FF C18:1n-9 and negatively with the proportions of C18:0 or C20:5n-3. TRIAL REGISTRATION: This study' registration number was B670201627735.

Human germline nuclear transfer to overcome mitochondrial disease and failed fertilization after ICSI.

PURPOSE: Providing additional insights on the efficacy of human nuclear transfer (NT). Here, and earlier, NT has been applied to minimize transmission risk of mitochondrial DNA (mtDNA) diseases. NT has also been proposed for treating infertility, but it is still unclear which infertility indications would benefit. In this work, we therefore additionally assess the applicability of NT to overcome failed fertilization. METHODS: Patient 1 carries a homoplasmic mtDNA mutation (m.11778G > A). Seventeen metaphase II (MII) oocytes underwent pre-implantation genetic testing (PGT), while five MII oocytes were used for spindle transfer (ST), and one in vitro matured (IVM) metaphase I oocyte underwent early pronuclear transfer (ePNT). Patients 2-3 experienced multiple failed intracytoplasmic sperm injection (ICSI) and ICSI-assisted oocyte activation (AOA) cycles. For these patients, the obtained MII oocytes underwent an additional ICSI-AOA cycle, while the IVM oocytes were subjected to ST. RESULTS: For patient 1, PGT-M confirmed mutation loads close to 100%. All ST-reconstructed oocytes fertilized and cleaved, of which one progressed to the blastocyst stage. The reconstructed ePNT-zygote reached the morula stage. These samples showed an average mtDNA carry-over rate of 2.9% ± 0.8%, confirming the feasibility of NT to reduce mtDNA transmission. For patient 2-3 displaying fertilization failure, ST resulted in, respectively, 4/5 and 6/6 fertilized oocytes, providing evidence, for the first time, that NT can enable successful fertilization in this patient population. CONCLUSION: Our study showcases the repertoire of disorders for which NT can be beneficial, to overcome either mitochondrial disease transmission or failed fertilization after ICSI-AOA.

Transgender men: clinical care and implications in reproductive medicine: introduction.

Gender dysphoria, a discrepancy between gender identity and genetically determined sex, is encountered in approximately 0.5% of people uniformly across the world. In the case of transgender men, formerly called female-to-male transsexuals, the available gender-affirming measures, hormone therapy and possible surgical procedures, are multiple and discussed in detail in this series of articles.

Ten years of fertility treatment experience and reproductive options in transgender men.

BACKGROUND: Up to 2018, the Belgian law stated that transgender people who wanted to change their legal sex had to undergo physical gender affirming treatment. This included gonadectomy to a medically possible and justified extent, which entailed that they had to accept the fact that they could no longer reproduce. However, research has shown that many transgender people desire to have children. AIMS: (1) to describe a cohort of transgender men and their respective cisgender female partners, to share our experiences with their request for donor conception, and to evaluate their disclosure intentions to the child, (2) to explore how the couples approach current and future reproductive options. METHODS: This mixed method study presents data from a retrospective analysis of patient records and from a qualitative interview study. The couples were selected from the group of transgender men who - together with their respective cisgender female partners - applied for sperm donation at Ghent University Hospital between 2002 and 2012. RESULTS: Forty-seven transgender men with a cisgender female partner requested treatment with anonymous donor sperm for a first child as a couple. Forty-one requests were accepted for treatment. We found that most couples requesting treatment intended to disclose the use of donor sperm to their future child (n = 34) while 24 couples were planning to inform the child about the parent's transgender identity. The six couples we interviewed saw donor conception as the preferred route to become parents. Adoption was seen as less obvious. The couples' attitudes toward stem cell-derived gametes reflected the significance of the genetic link with the child for both parents. DISCUSSION: Not all participants in our study were aware of their reproductive options. To be able to make a well-informed decision, transgender people should be counseled about all options at the time of transition.

Sex-Specific Isolation and Propagation of Human Premeiotic Fetal Germ Cells and Germ Cell-Like Cells.

The second trimester of human development is marked by asynchronous gonadal development hampering the isolation of homogenous populations of early and late fetal germ cells (FGCs). We evaluated the feasibility of using surface markers TNAP, PDPN, EPCAM and ITGA6 to isolate FGCs as well as human primordial germ cell-like cells (hPGCLCs) derived from embryonic stem cells (hESCs) from both sexes by fluorescence-activated cell sorting (FACS). Our results suggest that a combination of TNAP and PDPN was sufficient to separate populations of premeiotic FGCs and hPGCLCs in both sexes. This combination of antibodies also proved efficient in separating 'mitotic' from 'retinoic-acid responsive' female FGCs. Furthermore, we report that the differentiation efficiency of TNAP+PDPN+ hPGCLCs from hESCs was sex-independent, but the ability to propagate differed considerably between the sexes. In contrast to male, female hPGCLCs retained their characteristics and exhibited robust colony-forming ability when cultured for five days in medium containing LIF, forskolin and FGF2. We conclude that marked sex differences exist in the isolation and propagation of human FGCs and hPGCLCs. Our study provides novel insights relevant for the optimization of in vitro gametogenesis in humans.

Low feasibility of in vitro matured oocytes originating from cumulus complexes found during ovarian tissue preparation at the moment of gender confirmation surgery and during testosterone treatment for fertility preservation in transgender men.

OBJECTIVE: To study the feasibility of in vitro maturation of ovarian tissue oocytes for fertility preservation in transgender men on testosterone treatment. DESIGN: Cross-sectional study SETTING: University hospital PATIENT(S): Eighty-three transgender men enrolled from November 2015 to January 2019 INTERVENTION(S): In vitro maturation of cumulus-oocyte complexes (COCs) harvested at the time of gender confirmation surgery, and fertilization through intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): In vitro maturation, fertilization, and blastulation rates; comparison of morphokinetics with vitrified-warmed oocytes; and analysis of the genetic profiles of embryos. SECONDARY OUTCOMES: association between serum hormone levels; COCs' morphologic characteristics, and vitrification rate. RESULT(S): All participants were on testosterone treatment for a median of 83 (64[Quartile 1]; 113.2[Quartile 2]) weeks. A total of 1,903 COCs (mean per participant, 23 ± 15.8) were collected. The in vitro maturation rate was 23.8%, vitrification rate was 21.5%, and survival rate after warming was 72.6% (n = 151). Intracytoplasmic sperm injection was performed in 139 oocytes. The rate of normal fertilized oocytes was 34.5%, and 25 (52.1%) embryos reached day 3. One blastocyst was achieved on day 5. Aberrant cleavage patterns and early embryo arrest were observed in 22 (45.8%) and 44 (91.7%) zygotes, respectively. Compared with vitrified-warmed donor oocytes, a delay was observed in pronuclei disappearance, t2 (time to reach 2 cell stage) timings, and CC1 (the duration of the 1st cell cycle) and SS3 (synchronization of cleavage pattern (calculated as t8-t5) time intervals. A normal genetic pattern was seen in 42% embryos. The proportion of vitrified oocytes was negatively associated with progesterone (odds ratio, 0.76) and positively associated with antimüllerian hormone serum levels (odds ratio, 1.23). The highest vitrification rate was achieved by the morphologic characteristic 344 at day 0 and by 433 at day 2. CONCLUSION(S): Ovarian tissue oocytes matured in vitro show low developmental capacity in transgender men, when collected under testosterone treatment.

Activin A-derived human embryonic stem cells show increased competence to differentiate into primordial germ cell-like cells.

Protocols for specifying human primordial germ cell-like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post-inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i-state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP-2 gamma), SOX17 (SRY-box transcription factor 17), and NANOS3 (nanos C2HC-type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C-X-C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i-state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation.

Assessment of uterine activity during IVF by quantitative ultrasound imaging: a pilot study.

RESEARCH QUESTION: Does uterine activity differ in patients who have undergone successful IVF treatment compared with patients who have undergone unsuccessful IVF treatment? DESIGN: Prospective study of 16 women who underwent fresh single embryo transfer. All patients underwent transvaginal ultrasound in three phases of the IVF treatment: ovarian stimulation 1 h before embryo transfer (ET1) and 5-7 days after embryo transfer (ET5-7). Uterine motion analysis was implemented by a dedicated speckle tracking algorithm; frequency- and amplitude-related features were extracted from the derived signals to characterize the uterine activity in relation to ongoing implantation (positive HCG after 6 weeks) and ongoing pregnancy at 11 weeks. RESULTS: Uterine activity in terms of frequency (ovarian stimulation ET1, P = 0.04; ovarian stimulation ET5-7, P = 0.002) and amplitude (ovarian stimulation ET1, P = 0.0003; ovarian stimulation ET5-7, P = 0.000008) is significantly higher in the ovarian stimulation phase compared with ET1 and ET5-7. Women with ongoing pregnancies showed significantly higher uterine contraction frequency compared with those with no ongoing pregnancies in all phases (ovarian stimulation, P = 0.006; ET1, P = 0.015; ET5-7, P = 0.007). Uterine contraction amplitude was significantly lower (P = 0.037) in women at ET5-7 in women with ongoing pregnancies. CONCLUSIONS: This study is a first step towards assessing uterine activity during IVF objectively and non-invasively. It is an essential step to understanding the previously suggested effect of contractions on IVF failure. Uterine activity after embryo transfer characterized by high frequency and low amplitude may favour embryo implantation. Research with larger patient cohorts is needed to build on current evidence and knowledge of uterine contractions during IVF.

DSD: A Discussion at the Crossroads of Medicine, Human Rights, and Politics.

Over the past years, the topic of "disorder/differences in sex development (DSD)" or "intersex" people has become subject of the international political agenda. In 2017, a resolution of the Parliamentary Assembly of the Council of Europe argued that the practice of surgically modifying intersex children's genitals without medical necessity and without consent of the person concerned is a human rights violation. This resolution and related statements might impact heavily on pediatric urologists and their practice. While this resolution concerns a form of soft law and is not directly enforceable in member states, it might impact the national debates concerning legislation and medical guidelines on DSD. Consequently, this article reflects on this discussion by elaborating on the importance of human rights in our evolving understanding and legislation on DSD and other gender and sexuality issues in general. It constitutes a plea for a dialogue between medical professionals, lawmakers and human rights scholars which would lead to legislation and medical guidelines that take a holistic and rights-based approach.

The last vial. What it means to (aspiring) parents to use the same sperm donor for siblings.

Introduction: A lot of attention has been given to the quest of parents, children and donors to find donor siblings (= half siblings who share the same donor gametes but who are born in different families). However, literature is scarce about the use of the same sperm donor for subsequent children in the same family.Methods: This study included 68 lesbian and heterosexual (aspiring) parents, recruited at the Department of Reproductive Medicine of Ghent University Hospital (Belgium). The in-depth semi-structured couple interviews were performed between October 2012 and October 2013. Data were analyzed through step-by-step inductive thematic analysis.Results: The couples showed a clear preference to use the same sperm donor for their children. The most common reasons for this preference were related to the family or sibling relationships and medical reasons. Uncertainty about the availability of the same donor over time seeped through in their stories. Most lesbian aspiring parents decided that both partners should have a genetic link with at least one child.Conclusion: The use of the same sperm donor for subsequent conceptions appeared quasi unambiguously in the interviews of the lesbian and heterosexual (aspiring) parents in our study.

Donor insemination disclosure in social networks: heterosexual couples' experiences.

The way in which heterosexual couples manage information about infertility and donor insemination within their social networks has not yet been explored in-depth. This study focuses on how parents and aspiring parents manage information about infertility and donor insemination within their social networks. Fifteen Belgian couples were interviewed as part of a parenthood research project. Thematic analysis resulted in the identification of four themes. The first of these reveals how the social context can best be understood as a continuous confrontation with social expectations. A second theme highlights the diverse ways in which couples manage personal information in this confronting context. The third theme stresses how couples manage information about donor insemination so as to be treated as a 'normal' family. The final theme shows how emotional regulation within the context of the extended family plays a role in couples' decisions about how to manage information with relatives. Results are analysed using the concept of 'systemic emotion management' and the importance of being seen by others as a 'normal' family. Study findings signal the importance of managing information within social networks and are of relevance to a range of practitioners.

Impaired implantation in endometriosis compared with couples with male subfertility after transfer of equal quality embryos: a matched cohort study.

RESEARCH QUESTION: Is implantation impaired in patients with endometriosis undergoing IVF and intracytoplasmatic sperm injection (ICSI) cycles? DESIGN: A retrospective matched cohort study was carried out on IVF/ICSI cycles with fresh single embryo transfer at the Department of Assisted Reproductive Medicine, Ghent University Hospital, Belgium, between July 2015 and August 2017 (n = 1053). A total of 118 endometriosis cases were matched 1:1 to 118 couples diagnosed with male subfertility and stratified by embryo quality (identical ALPHA grading categories), female age (±1 year) and parity (±1 delivery). Transvaginal ultrasound, magnetic resonance imaging or laparoscopy was used to diagnosed endometriosis, and the revised American Society for Reproductive Medicine score was used to classify the endometriosis into grade I/II versus grade III/IV. Male subfertility was defined in accordance with World Health Organization criteria (fifth edition). RESULTS: Compared with endometriosis cases, control couples with male subfertility had significantly higher rates of positive HCG test on day 16 (P = 0.047, OR 2.077, CI 1.009 to 4.276), ongoing implantation (defined as a positive fetal heart rate on transvaginal ultrasound at a gestational age of at least 6.5-7 weeks) (P = 0.038, OR 2.265, CI 1.048 to 4.893), ongoing pregnancy (defined by a vital pregnancy at 11 weeks) (P = 0.046, OR 2.292, CI 1.016 to 5.173) and live birth (P = 0.043, OR 2.502, CI 1.029 to 6.087). CONCLUSIONS: After matching for embryo quality, woman's age and parity, rates of positive HCG tests, ongoing implantation, ongoing pregnancy and live birth were more than twice as high in the control group compared with the endometriosis group.

In-vitro development of embryos derived from vitrified-warmed oocytes is delayed compared with embryos derived from fresh oocytes: a time-lapse sibling oocyte study.

RESEARCH QUESTION: Is there a difference in blastocyst formation between fresh and vitrified-warmed sibling oocytes and can this difference be attributed to changes in embryo morphokinetics? DESIGN: Between February 2016 and December 2017, 472 metaphase II (MII) oocytes in 67 donor-recipient cycles from 27 different healthy anonymous oocyte donors were allocated for fresh transfer (FSHO) (n = 220) to a synchronous recipient (n = 36) or vitrified (VITO) (n = 252) to be warmed and transferred to another recipient (n = 31). Embryos derived from the FSHO and their sibling VITO were analysed for morphokinetic development using time-lapse imaging, blastocyst formation and clinical outcome. RESULTS: Time-lapse analysis showed an overall delay in cleavage rate from the time of pronuclei disappearance up to the time of blastulation in the VITO compared with their sibling FSHO. Twelve morphokinetic variables were significantly different between the groups. On Day 5 significantly more FSHO embryos developed to blastocyst (expansion 1-6) and reached the full blastocyst stage (expansion 3-6) compared with the VITO embryos [53.2% (84/158) versus 40.0% (64/160); P = 0.0244 and 48.1% (76/158) versus 31.3% (50/160); P = 0.0028, respectively]. The embryo utilization rate was similar in both groups at the time of cryopreservation; 51.3% (FSHO) versus 45.0% (VITO) (P = 0.3124). The pregnancy rate per cycle was 47.2% (17/36) in FSHO patients and 48.4% (15/31) in VITO patients (P = 1). Limitations in this study: non-randomized, small study size and not powered to detect differences in clinical outcomes. CONCLUSIONS: Timing of development is altered and blastocyst formation is delayed in embryos derived from vitrified-warmed donor oocytes compared with their fresh sibling counterparts. Although preliminary results suggest that the clinical impact of this delay may be limited, this needs further investigation in larger randomized studies.

Vitrification negatively affects the Ca2+-releasing and activation potential of mouse oocytes, but vitrified oocytes are potentially useful for diagnostic purposes.

RESEARCH QUESTION: To what extent does vitrification affect the Ca2+-releasing and activation potential of mouse oocytes, which are commonly used to determine the oocyte activation potential of human spermatozoa? DESIGN: The effect of mouse oocyte vitrification on Ca2+ dynamics and developmental competence after oocyte activation was assessed and compared with fresh mouse oocytes. Moreover, the Ca2+ store content of the endoplasmic reticulum was determined at different time points during the vitrification-warming procedure. Finally, the Ca2+ pattern induced by cryoprotectant exposure was determined. RESULTS: After human sperm injection into mouse oocytes, Ca2+ dynamics but not fertilization rates were significantly altered by vitrification warming (P < 0.05). Ca2+ dynamics in response to SrCl2 or ionomycin were also altered by oocyte vitrification. In contrast, activation and blastocyst rates after SrCl2 exposure were not affected (P > 0.05), whereas activation rates after ionomycin exposure were significantly lower in vitrified-warmed oocytes (P < 0.05); blastocyst rates were not affected (P > 0.05). Cryoprotectant exposure was associated with a strong drop in endoplasmic reticulum Ca2+ store content. Oocytes rapidly recovered during warming and recovery in Ca2+-containing media; a threshold area under the curve of Ca2+ dynamics to obtain activation rates above 90% was determined. CONCLUSIONS: Vitrified-warmed mouse oocytes display reduced Ca2+-releasing potential upon oocyte activation, caused by cryoprotectant exposure. With adapted classification criteria, these oocytes could be used for diagnosing oocyte activation deficiencies in patients. Evaluating the Ca2+-signalling machinery in vitrified-warmed human oocytes is required.

Untargeted histone profiling during naive conversion uncovers conserved modification markers between mouse and human.

Recent progress has enabled the conversion of primed human embryonic stem cells (hESCs) to the naive state of pluripotency, resembling the well-characterized naive mouse ESCs (mESCs). However, a thorough histone epigenetic characterization of this conversion process is currently lacking, while its likeness to the mouse model has not been clearly established. Here, we profile the histone epigenome of hESCs during conversion in a time-resolved experimental design, using an untargeted mass spectrometry-based approach. In total, 23 histone post-translational modifications (hPTMs) changed significantly over time. H3K27Me3 was the most prominently increasing marker hPTM in naive hESCs. This is in line with previous reports in mouse, prompting us to compare all the shared hPTM fold changes between mouse and human, revealing a set of conserved hPTM markers for the naive state. Principally, we present the first roadmap of the changing human histone epigenome during the conversion of hESCs from the primed to the naive state. This further revealed similarities with mouse, which hint at a conserved mammalian epigenetic signature of the ground state of pluripotency.

Human blastocyst outgrowths recapitulate primordial germ cell specification events.

Our current knowledge of the mechanisms leading to human primordial germ cell (PGC) specification stems solely from differentiation experiments starting from human pluripotent stem cells. However, information regarding the origin of PGCs in vivo remains obscure. Here we apply an improved system for extended in vitro culture of human embryos to investigate the presence of PGC-like cells (PGCLCs) 12 days post fertilization (dpf). Good quality blastocysts (n = 141) were plated at 6 dpf and maintained in hypoxia, in medium supplemented with Activin A until 12 dpf. We primarily reveal that 12 dpf outgrowths recapitulate human peri-implantation events and demonstrate that blastocyst quality significantly impacts both embryo viability at 12 dpf, as well as the presence of POU5F1+ cells within viable outgrowths. Moreover, detailed examination of 12 dpf blastocyst outgrowths revealed a population of POU5F1+, SOX2- and SOX17+ cells that may correspond to PGCLCs, alongside POU5F1+ epiblast-like cells and GATA6+ endoderm-like cells. Our findings suggest that, in human, PGC precursors may become specified within the epiblast and migrate either transiently to the extra-embryonic mesoderm or directly to the dorsal part of the yolk sac endoderm around 12 dpf. This is a descriptive analysis and as such the conclusion that POU5F1+ and SOX17+ cells represent bona fide PGCs can only be considered as preliminary. In the future, other PGC markers may be used to further validate the observed cell populations. Overall, our findings provide insights into the origin of the human germline and may serve as a foundation to further unravel the molecular mechanisms governing PGC specification in human.