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Autologous endometrial cell co-culture improves human embryo development to high-quality blastocysts: a randomized controlled trial

  • Author Footnotes
    † These authors contributed equally to this work.
    Cécile Le Saint
    Correspondence
    Corresponding authors.
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Clinique Ovo, Ovo Labo, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada
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  • Author Footnotes
    † These authors contributed equally to this work.
    Kimberley Crespo
    Correspondence
    Corresponding authors.
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Clinique Ovo, Ovo Labo, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada
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  • Amélie Bourdiec
    Affiliations
    Clinique Ovo, Ovo R and D, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada
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  • François Bissonnette
    Affiliations
    Clinique Ovo, Ovo Labo, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    Clinique Ovo, Ovo R and D, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    Clinique Ovo, Ovo Fertility, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    University of Montréal, Department of Obstetrics and Gynecology, Saint-Luc Hospital (CHUM), 1051 Sanguinet Street, Montréal Quebec H2 × 0C1, Canada
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  • Karen Buzaglo
    Affiliations
    Clinique Ovo, Ovo Fertility, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    University of Montréal, Department of Obstetrics and Gynecology, Saint-Luc Hospital (CHUM), 1051 Sanguinet Street, Montréal Quebec H2 × 0C1, Canada
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  • Author Footnotes
    † These authors contributed equally to this work.
    Bernard Couturier
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Clinique Ovo, Ovo Labo, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    Clinique Ovo, Ovo R and D, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    Clinique Ovo, Ovo Fertility, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    University of Montréal, Department of Obstetrics and Gynecology, Saint-Luc Hospital (CHUM), 1051 Sanguinet Street, Montréal Quebec H2 × 0C1, Canada
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  • Sandra Bisotto
    Affiliations
    Clinique Ovo, Ovo R and D, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada
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  • Simon J. Phillips
    Affiliations
    Clinique Ovo, Ovo Fertility, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    Faculty of Medicine, University of Montréal, Montréal Quebec, Canada
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  • Melissa Stutz
    Affiliations
    JSS Research, 9400 Henri-Bourassa West, St-Laurent Quebec H4S 1N8, Canada
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  • Jean-Noël Gouze
    Affiliations
    Laboratoires Genévrier, 280 de Goa Street, ZI des Trois Moulins Antibes 06600, France
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  • John S. Sampalis
    Affiliations
    JSS Research, 9400 Henri-Bourassa West, St-Laurent Quebec H4S 1N8, Canada
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  • Samir Hamamah
    Affiliations
    ART/PGD Department, Arnaud-de-Villeneuve Hospital, University Hospital of Montpellier, Inserm U1203, CHRU Montpellier, Montpellier 34295, France
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  • Isaac Jacques Kadoch
    Affiliations
    Clinique Ovo, Ovo Labo, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    Clinique Ovo, Ovo R and D, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    Clinique Ovo, Ovo Fertility, 8000 Décarie Boulevard, Suite 600, Montréal Quebec H4P 2S4, Canada

    University of Montréal, Department of Obstetrics and Gynecology, Saint-Luc Hospital (CHUM), 1051 Sanguinet Street, Montréal Quebec H2 × 0C1, Canada
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  • Author Footnotes
    † These authors contributed equally to this work.
Published:January 05, 2019DOI:https://doi.org/10.1016/j.rbmo.2018.12.039

      Abstract

      Research question

      Does autologous endometrial cell co-culture (AECC) improve the number of good-quality blastocysts obtained by IVF/intracytoplasmic sperm injection (ICSI), compared with conventional embryo culture medium in a broad group of patients referred to assisted reproductive technology (ART)?

      Design

      This interventional, randomized, double-blind study took place at Clinique Ovo from March 2013 to October 2015 and included 207 healthy patients undergoing an IVF or ICSI protocol, of which 71 were excluded before randomization. On the previous cycle, all participants underwent an endometrial biopsy at D5 to D7 post-ovulation, following which the endometrial cells were prepared for AECC.

      Results

      The data demonstrated that AECC significantly increased the incidence of good-quality blastocysts compared with culture in conventional media (42.6% vs 28.4%, P < 0.001). No significant differences were found in pregnancy and live birth rates.

      Conclusion

      This study demonstrated the benefits of AECC on blastocyst quality compared with conventional embryo culture medium, in a broader category of patients referred to ART as opposed to other studies that concentrated on specific causes of infertility only. However, limitations of the study design should be taken into consideration; the analysis was performed using embryos rather than patients and a follow-up of children born following the treatments could not be conducted.

      Keywords

      Introduction

      One of the key factors in achieving pregnancies in assisted reproductive technologies (ART) is maximizing embryo quality, which is heavily influenced by embryo culture conditions.
      In this context, several strategies have been proposed in order to improve embryo culture conditions and pregnancy rates (
      • Swain J.E.
      • Carrell D.
      • Cobo A.
      • Meseguer M.
      • Rubio C.
      • Smith G.D.
      Optimizing the culture environment and embryo manipulation to help maintain embryo developmental potential.
      ). Many studies have provided evidence related to the impact of culture conditions on the success of IVF and the development of the future offspring, leading to the categorization of embryo culture media as a class III medical device in the European Union and a class II in North America (
      • Chronopoulou E.
      • Harper J.C.
      IVF culture media: Past, present and future.
      ,
      • Kleijkers S.H.M.
      • Eijssen L.M.T.
      • Coonen E.
      • Derhaag J.G.
      • Mantikou E.
      • Jonker M.J.
      • Mastenbroek S.
      • Repping S.
      • Evers J.L.H.
      • Dumoulin J.C.M.
      • van Montfoort A.P.A.
      Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media.
      ,
      • Mantikou E.
      • Youssef M.A.F.M.
      • van Wely M.
      • van der Veen F.
      • Al-Inany H.G.
      • Repping S.
      • Mastenbroek S.
      Embryo culture media and IVF/ICSI success rates: a systematic review.
      ). The first embryo culture media used were not optimized for blastocyst development. However, sequential media were developed with the purpose of mimicking in-vivo conception and the natural environment of the embryo, which changes in the course of its development, comprising physiological fluids such as tubal or uterine fluid (
      • Chronopoulou E.
      • Harper J.C.
      IVF culture media: Past, present and future.
      ). With the emergence of time-lapse imaging, which allows embryo development to be monitored in real time, single-step media gained popularity (
      • Chronopoulou E.
      • Harper J.C.
      IVF culture media: Past, present and future.
      ,
      • Meseguer M.
      • Rubio I.
      • Cruz M.
      • Basile N.
      • Marcos J.
      • Requena A.
      Embryo incubation and selection in a time-lapse monitoring system improves pregnancy outcome compared with a standard incubator: A retrospective cohort study.
      ). In essence, single-step media is supposed to contain all the nutrients that the embryo needs during its early development (
      • Meseguer M.
      • Pellicer A.
      The most well kept secret, embryo culture media : a smart reveal from an expert.
      ,
      • Meseguer M.
      • Rubio I.
      • Cruz M.
      • Basile N.
      • Marcos J.
      • Requena A.
      Embryo incubation and selection in a time-lapse monitoring system improves pregnancy outcome compared with a standard incubator: A retrospective cohort study.
      ). As detailed formulations are not provided by manufacturers, Morbeck et al. studied and compared several culture media, showing that they vary considerably in terms of composition (
      • Menezo Y.
      • Lichtblau I.
      • Elder K.
      New insights into human pre-implantation metabolismin vivo andin vitro.
      ,
      • Morbeck D.E.
      • Krisher R.L.
      • Herrick J.R.
      • Baumann N.A.
      • Matern D.
      • Moyer T.
      Composition of commercial media used for human embryo culture.
      ,
      • Morbeck D.E.
      • Paczkowski M.
      • Fredrickson J.R.
      • Krisher R.L.
      • Hoff H.S.
      • Baumann N.A.
      • Moyer T.
      • Matern D.
      Composition of protein supplements used for human embryo culture.
      a,
      • Morbeck D.E.
      • Paczkowski M.
      • Fredrickson J.R.
      • Krisher R.L.
      • Hoff H.S.
      • Baumann N.A.
      • Moyer T.
      • Matern D.
      Composition of protein supplements used for human embryo culture.
      b). Furthermore, several studies using different sources of cells in their media preparations reported an improvement in terms of embryo quality and blastulation rate (
      • Feng H.L.
      • Wen X.H.
      • Amet T.
      • Presser S.C.
      Effect of different co-culture systems in early human embryo development.
      ,
      • Gandolfi F.
      • Moor R.M.
      Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells.
      ,
      • Wiemer K.E.
      • Cohen J.
      • Amborski G.F.
      • Wright G.
      • Wiker S.
      • Munyakazi L.
      • Godke R.
      In-vitro development and implantation of human embryos following culture on fetal bovine uterine fibroblast cells.
      ).
      Several contradictory studies comparing neonatal outcomes such as gestational term, birth weight and congenital malformations between blastocyst stage versus early cleavage stage embryo IVF transfers seemed to be in favour of early cleavage stage transfers. Several of these studies actually showed a negative impact of the embryo's longer incubation in culture medium (
      • Marianowski P.
      • Dabrowski FA.
      • Zygula A.
      • Wielgos M.
      • Szymusik I.
      Do We Pay Enough Attention to Culture Conditions in Context of Perinatal Outcome afterIn vitro Fertilization ? Up-to-Date Literature Review.
      ). However, other key opinion leaders (KOL) provided evidence that blastocyst stage transfer leads to a higher rate of clinical pregnancy and live birth (
      • Papanikolaou E.
      • Kolibianakis E.
      • Tournaye H.
      • Venetis C.
      • Fatemi H.
      • Tarlatzis B.
      • Devroey P.
      Live birth rates after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and meta-analysis.
      ,
      • Schwärzler P.
      • Zech H.
      • Auer M.
      • Pfau K.
      • Gobel G.
      • Vanderzwalmen P.
      • Zech N.
      Pregnancy outcome after blastocyst transfer as compared with early cleavage stage embryo transfer.
      ). Thereby, in order to better support the development of early embryos into blastocysts by simulating the in-vivo environment, KOL developed the co-culture of human embryos (
      • Bolton V.N.
      • Cutting R.
      • Clarke H.
      • Brison D.R.
      ACE consensus meeting report: Culture systems.
      ,
      • Ménézo YJR.
      Blastocyst freezing.
      ,
      • Ohl J.
      • De Mouzon J.
      • Nicollet B.
      • Benoussaidh A.
      • Gouze J.
      • Viville S.
      • Menezo Y.
      Increased pregnancy rate using standardized coculture on autologous endometrial cells and single blastocyst transfer: A multicentre randomized controlled trial.
      ).
      Mimicking natural physiological processes, transfers of embryos at the blastocyst stage permit coordination of the dialogue between the embryo and the endometrial cells during the implantation window of the patient undergoing IVF treatment, and thus increases the chance of pregnancy (
      • Gardner D.K.
      • Lane M.
      Alleviation of the ‘2-cell block’ and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters.
      ,
      • Simón C.
      • Mercader A.
      • Garcia-Velasco J.
      • Nikas G.
      • Moreno C.
      • Remohí J.
      • Pellicer A.
      Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure.
      ).
      Co-culture systems, originally developed in the 1990s, used several cell types from non-human cells to human reproductive cells (
      • Simón C.
      • Mercader A.
      • Garcia-Velasco J.
      • Nikas G.
      • Moreno C.
      • Remohí J.
      • Pellicer A.
      Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure.
      ). Consequently, in 2002, the FDA limited the use of co-culture in human IVF treatments in order to avoid the risk of disease transmission from non-human or heterologous human cell lines (
      • Swain J.E.
      • Carrell D.
      • Cobo A.
      • Meseguer M.
      • Rubio C.
      • Smith G.D.
      Optimizing the culture environment and embryo manipulation to help maintain embryo developmental potential.
      ). Rather, autologous endometrial co-culture (AECC) was developed, using the patient's own endometrial cells.
      The establishment of autocrine and paracrine communications and cell-to-cell interactions between the endometrial feeder cells and the embryo should contribute to the detoxification of the culture medium, facilitating blastocyst development and improving the implantation rate (
      • Bochev I.
      • Belemezova K.
      • Shterev A.
      • Kyurkchiev S.
      Effect of cryopreservation on the properties of human endometrial stromal cells used in embryo co-culture systems.
      ,

      De los Santos, M., Mercader, A., Francés, A., Portoles, E., Remohí, J., Pellicer, A., Simón, C.Role of Endometrial Factors in Regulating Secretion of Components of the Immunoreactive Human Embryonic Interleukin-1 System during Embryonic Development. Biology of Reproduction. 1996; 54: 563–574

      ,
      • Guérin P.
      • El Mouatassim S.
      • Ménézo Y.
      Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings.
      ,
      • Mercader A.
      • Valbuena D.
      • Simón C.
      Human Embryo Culture.
      ,
      • Simón C.
      • Mercader A.
      • Garcia-Velasco J.
      • Nikas G.
      • Moreno C.
      • Remohí J.
      • Pellicer A.
      Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure.
      ). Many teams concentrating their studies more specifically on patients with IVF failure such as poor ovarian reserve, history of poor-quality embryos or repeated implantation failure, demonstrated the benefits of AECC in in-vitro treatments (
      • Eyheremendy V.
      • Raffo F.G.E.
      • Papayannis M.
      • Barnes J.
      • Granados C.
      • Blaquier J.
      Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure.
      ,
      • Spandorfer S.
      • Barmat L.
      • Navarro J.
      • Burmeister L.
      • Veeck L.
      • Clarke R.
      • Liu H.
      • Rosenwaks Z.
      Autologous endometrial coculture in patients with a previous history of poor quality embryos.
      ,
      • Spandorfer S.
      • Pascal P.
      • Parks J.
      • Clark R.
      • Veeck L.
      • Davis O.
      • Rosenwaks Z.
      Autologous endometrial coculture in patients with IVF failure: outcome of the first 1,030 cases.
      ). Recently a European, multicentre, prospective and randomized controlled trial assessed the efficacy of AECC, comparing the pregnancy rate after single embryo transfer of D5 blastocysts on AECC to a D3 embryo transfer, cultured in a conventional medium (
      • Ohl J.
      • De Mouzon J.
      • Nicollet B.
      • Benoussaidh A.
      • Gouze J.
      • Viville S.
      • Menezo Y.
      Increased pregnancy rate using standardized coculture on autologous endometrial cells and single blastocyst transfer: A multicentre randomized controlled trial.
      ). Intermediate analysis of this study showed that AECC improved early embryo and blastocyst quality and significantly increased the clinical pregnancy rate per transfer (
      • Ohl J.
      • De Mouzon J.
      • Nicollet B.
      • Benoussaidh A.
      • Gouze J.
      • Viville S.
      • Menezo Y.
      Increased pregnancy rate using standardized coculture on autologous endometrial cells and single blastocyst transfer: A multicentre randomized controlled trial.
      ). However, this study compared blastocysts subjected to co-culture with early cleavage stage embryos grown in conventional medium, hence limiting the scope of the given conclusion. Based on the hypothesis that all women undergoing IVF/intracytoplasmic sperm injection (ICSI) can and should benefit from AECC, the present study aimed to validate the efficacy of a single blastocyst (D5 or D6) transfer co-cultured on AECC compared to in a conventional medium in a cohort including a broad category of women.

      Materials and methods

      Study design

      This was an interventional, single-site, randomized, double-blind controlled study evaluating the benefits of AECC versus conventional medium in IVF or ICSI treatments.
      Embryo quality and blastulation rate were evaluated as primary endpoints comparing D5/D6 blastocyst transfer rates between the AECC and conventional medium groups. Biochemical and clinical pregnancy rates and live birth rates were then analysed as secondary endpoints.
      In order to detect a 10% increase in the rate of usable embryos (namely the embryos of sufficient quality to be transferred or vitrified) in the AECC versus conventional medium groups, 770 D2 embryos were required for inclusion, based on an average of 7 embryos per patient at Clinique Ovo (Montréal, Quebec, Canada). Factoring in a 15% dropout rate, a minimum of 128 patients were required, or 64 patients per group.

      Ethics statement

      This trial, approved by an independent ethics committee on 14 December 2012 (reference: OVO-12-24), was registered at ClinicalTrials.gov (NCT01886118) and was conducted in accordance with the Declaration of Helsinki. Informed consents were obtained from all participants prior to the performance of any study-related procedures.

      Participants

      Between April 2013 and March 2015, 207 healthy women aged between 18 and 38, undergoing an IVF or ICSI protocol with ovarian stimulation at Clinique Ovo, were screened for eligibility. Inclusion criteria included normal uterine cavity, regular menstrual cycles, basal FSH levels <10 IU/l, anti-Müllerian hormone (AMH) >1 ng/ml and/or antral follicle count (AFC) ≥12 within 12 months prior to entering the study and could not have previously undergone more than three IVF cycles with embryo transfer. The following patients were excluded: aged ≥39; amenorrhoea; anovulatory cycles; menstrual cycle >40 days; chronic endometritis; severe endometriosis (stages III and IV); hydrosalpinx; Asherman's syndrome or uterine synechia; uterine polyp, fibroids or other uterine anomalies; use of anticoagulant; male partner with secretory azoospermia and multiple embryo transfer.
      Based on the inclusion and exclusion criteria above, 136 patients were randomized to receive either AECC (n = 63) or conventional medium (n = 73).

      Endometrial biopsy

      For all enrolled participants, an endometrial biopsy was performed and treated in accordance with the Endocell® AECC protocol (Laboratoires Genévrier, Sophia Antipolis, Antibes, France). Briefly, prior to the IVF/ICSI stimulation cycle, participants were asked to perform basal body temperature curve monitoring as well as urinary LH testing (First Response; Church and Dwight Canada Corp., Mississauga, Ontario, Canada). Between D5 and D7 following ovulation (D7 to D9 following a positive urinary LH test), participants underwent an endometrial biopsy at Clinique Ovo with Pipelle de Cornier® endometrial sampling (Surgi-Pharm Avancee Inc., Canada). The biopsy was then prepared for cryopreservation using the provided Endocell® kit, according to protocols and procedures from Laboratoires Genévrier. All participants were tested for Mycoplasma hominis, Chlamydia trochomatis and Ureaplasma urealyticum by vaginal swab prior to the biopsy. Patients with positive results were treated with antibiotics; these patients continued their IVF cycle but were excluded from the present study.

      Ovarian stimulation protocols

      Gonadotrophins were prescribed at the physician's discretion between the gonadotrophin-releasing hormone (GnRH) antagonist, short GnRH agonist or SMART (stimulation with minimal adverse effects, retrieval and transfer) protocols, according to the age and ovarian reserve test results (AMH, FSH and AFC). The FSH doses were then adjusted according to follicular growth until the day of ovulation triggering by the human chorionic gonadotrophin (HCG) hormone (5000 IU) (Ferring Canada, North York, Canada).

      Randomization

      Participants were randomized 1:1 to either the AECC group (study group) or conventional medium group (control group) by opening a randomization envelope on the day of HCG administration, 36 h prior to oocyte retrieval. Randomization was performed using an Excel file. Only the research associates and the embryologists were aware of the randomization group; physicians and participants were kept blinded until all vitrified embryos were transferred or the end of the study. In the case of a negative pregnancy test, all patients randomized in the conventional medium group were offered the option to use AECC in their subsequent treatment attempt.

      Autologous endometrial co-culture

      Endometrial tissue was kept in liquid nitrogen until the participant's randomization. The day prior to oocyte retrieval, endometrial tissue was carefully thawed in order to proceed to cell preparation for the endometrial culture. All solutions were provided in the Endocell® kit (Laboratoires Genévrier). Briefly, cryovials were warmed in a 37°C water bath. Tissue was then processed to isolate epithelial and stromal cellular fractions. Endometrial cells were then seeded in a monolayer, on IVF grade tissue culture plates (Corning, USA), in a 1/75 epithelial gland/stromal cell ratio. Cells were cultured in CCM-30 (Vitrolife Inc., CO, USA) and incubated at 37°C with 5% CO2 and 20% O2. After in-vitro oocyte fertilization either by regular IVF or ICSI, D2 healthy developing embryos were transferred, one per well containing 1 ml blastocyst culture medium, over the endometrial culture, to pursue their development until D5 or D6. A maximum of eight embryos were placed in co-culture due to restrictions in terms of the plate's dimension: the first eight embryos were arbitrarily assigned to co-culture, in a non-selective way. Hence, supernumerary embryos from the study group were placed in the conventional medium. Blastocysts qualifying as high quality were either transferred on D5 or vitrified for future use and good-quality blastocysts on D6 were vitrified.

      Early embryo culture in conventional media

      Fertilized oocytes confirmed by the presence of two pronuclei were cultured in 20 μl drops of Global Total (Global, Toronto, Canada) under oil (Global) in 35 mm Falcon Primaria™ dishes (Fisher Scientific, Canada). Embryos were assessed daily for development, except D4, and media was changed at the end of D2. Embryos were cultured in IVF-specific incubators (G185, K-Systems, Denmark) at 37°C, 6% CO2 and 5% O2. Blastocyst assessment was carried out on D5 and D6 using the modified Gardner's system (Veeck et al., 2003) and blastocysts of BB score or higher were considered for transfer or vitrification (
      • Liebermann J.
      Chapter 11 Human Embryo Vitrification.
      ).

      Embryo grading

      Embryos were scored according to the criteria described in the
      ALPHA Scientists in Reproductive Medicine, ESHRE Special Interest Group Embryology
      Istanbul consensus workshop on embryo assessment: proceeding of an Expert Meeting.
      . Transferable embryos were defined on D3 as having between 6 and 8 cells and grade 2 or better. For D5 and D6, usable blastocysts were defined as blastocysts with a BB score or higher. The blastocysts obtained were either transferred fresh into the uterus at D5 or vitrified for further transfer.

      Pregnancy outcomes

      Biochemical pregnancy was determined by serum β-HCG test and clinical pregnancy was determined by the presence of a fetal heart visualized on ultrasound at 8 weeks. A miscarriage was defined with a positive β-HCG test but no fetal heart activity observed on ultrasound, as well as clinical pregnancies that do not result in a live birth.

      Statistical analysis

      Descriptive statistics were produced for all study variables, including the mean, standard deviation, and 95% confidence interval (CI) for continuous variables, and frequency distributions for categorical variables. Between-group differences with respect to demographic characteristics, oocyte characteristics and IVF/ICSI outcomes were assessed using one-way analysis of variance (ANOVA), the chi-squared test or Fisher's exact test, as appropriate.
      Multivariate logistic regression was used to ascertain predictors of fertilization, blastulation and pregnancy outcomes for multiple covariates such as smoking, infertility causes, IVF rank protocol types, stimulation days, total number of oocytes and number of metaphase II (MII) oocytes retrieved (Table 1). Within each group, pregnancy outcomes were stratified by fresh versus vitrified-warmed embryo transfer.
      Table 1Baseline demographic and cycle characteristics
      ParameterGroup
      AECC
      Baseline characteristics assessed in randomized patients.
      CM
      Baseline characteristics assessed in randomized patients.
      Demographicsn = 63n = 73
      Age, female, years (mean ± SD)32.8 ± 3.532.6 ± 4.2
      Age, male, years (mean ± SD)36.6 ± 5.935.7 ± 9.7
      BMI, kg/m2 (mean ± SD)23.9 ± 5.324.2 ± 5.4
      Smoker, yes, n (%)5 (7.9)10 (13.7)
      Infertility cause
       Female factor, n (%)20 (31.7)18 (24.7)
       Male factor, n (%)20 (31.7)27 (37.0)
       Mixed, n (%)6 (9.5)9 (12.3)
       Idiopathic, n (%)17 (27.0)19 (26.0)
      IVF/ICSI attempt number
       1, n (%)32 (50.8)46 (63)
       2, n (%)20 (31.7)19 (26)
       3, n (%)8 (12.7)5 (6.8)
       4, n (%)2 (3.2)2 (2.7)
       5, n (%)1 (1.6)1 (1.4)
      Oocytesn = 56n = 70
      Stimulation, days (mean ± SD)11.6 ± 1.411.6 ± 1.4
      Number of oocytes collected (mean ± SD)13.5 ± 5.814.1 ± 8.2
      Number of metaphase II oocytes (mean ± SD)10.1 ± 4.610.7 ± 5.8
      Number of fertilized oocytes (mean ± SD)6.8 ± 4.36.6 ± 4.3
      Stimulation protocol
       Antagonist GnRH, n (%)56 (88.9)69 (94.5)
       Agonist GnRH, n (%)7 (11.1)3 (4.1)
       SMART, n (%)0 (0.0)1 (1.4)
      No significant between-group differences were identified using one-way analysis of variance (ANOVA) and the chi-squared test for continuous and categorical variables, respectively.
      95% CI = 95% confidence interval for mean; AECC = autologous endometrial co-culture group; BMI = body mass index; CM = conventional medium group; GnRH = gonadotrophin-releasing hormone; ICSI = intracytoplasmic sperm injection; SMART = stimulation with minimal adverse effects, retrieval and transfer.
      a Baseline characteristics assessed in randomized patients.
      Furthermore, a predictive model was constructed in order to ascertain the probability of biochemical pregnancy, clinical pregnancy and live birth in the remaining vitrified embryos.
      Statistical analysis was performed using IBM SPSS Statistics (version 24). Statistical significance was accepted when P < 0.05.

      Results

      Participant flow

      A participant flow chart is presented in Figure 1. From 207 patients enrolled in the study, 71 were excluded before randomization due to either failure in ovulation detection using urinary LH detection kit (n = 20), cancelled stimulation cycle (n = 7), spontaneous pregnancy (n = 4), personal reasons (n = 19) and other reasons (n = 21). A total of 1406 mature oocytes were recovered from the 136 randomized patients. These oocytes had been fertilized either by classical IVF or ICSI. Of these mature oocytes, a total of 507 were withdrawn from consideration in the pregnancy outcome analyses due to unsuccessful fertilization (n = 475) or double embryo transfer (n = 32). Because the AECC group could only allow a maximum of 8 embryos per participant due to restrictions from the Endocell® culture plates, the supernumerary embryos (n = 115) were placed in the conventional medium (control group) and analysed as controls. The study group (AECC) was then composed of 326 embryos co-cultured on autologous endometrial cells from D2. The remaining 115 supernumerary embryos initially randomized in the AECC group were then added to the 458 control group embryos and were grown in conventional medium following Clinique Ovo's standard culture protocols (Figure 1).
      Figure 1
      Figure 1Participant flow chart. Embryos included in double embryo transfers were excluded. aBecause the AECC group could only allow a maximum of eight embryos per participant due to restriction from the culture plates’ dimension, the supernumerary embryos (n = 115) were placed in the conventional medium (control group) and analysed as controls. AECC group = autologous endometrial co-culture group; CM group = conventional medium group; FET = frozen embryo transfers.
      Embryos that did not develop according to the defined criteria were discarded (n = 176 in the study group vs n = 388 in the control group). Among the quality D3 embryos and blastocysts obtained from the two groups, one selected embryo per participant was transferred in a fresh embryo transfer (n = 40 in the study group vs n = 42 in the control group). Supernumerary good embryos were vitrified for further use. One hundred and ten (110) early cleavage stage embryos and blastocysts were vitrified in the AECC group. Of these, 41 were transferred after warming, 7 degenerated post-warming and 62 were still cryopreserved at the time of the analysis. In the conventional medium group, from the 143 vitrified embryos, 39 were transferred; 16 degenerated post-warming and 88 were still cryopreserved (Figure 1).

      Baseline characteristics

      Population homogeneity between the two studied groups was assessed with the results provided in Table 1. Patient demographic characteristics, assessed in randomized patients, found between-group similarity with respect to patient age (32.8 ± 3.5 and 32.6 ± 4.2 years in the AECC and control group, respectively), partner age (36.6 ± 5.9 years vs 35.7 ± 9.7 years) and body mass index (23.9 ± 5.3 kg/m2 vs 24.2 ± 5.4 kg/m2). In addition, no significant between-group differences were observed between oocyte characteristics with respect to stimulation duration (11.6 ± 1.4, AECC and control group, respectively), number of collected oocytes (13.5 ± 5.8 vs 14.1 ± 8.2 in the AECC and control groups, respectively), and number of MII (10.1 ± 4.6 and 10.7 ± 5.8) and fertilized oocytes (6.8 ± 4.3 vs 6.6 ± 4.3) (Table 1).
      No between-group differences in smoking status, infertility cause, IVF/ICSI attempt number or stimulation protocol were observed (Table 1). Ninety-two per cent (92%) of the participants were treated using an antagonist protocol; 7% followed a short agonist protocol. Sixty-three per cent (63%) of the patients had their oocytes fertilized by ICSI, 26% by classical IVF, 11% had half of their oocytes fertilized by ICSI and half by classical IVF.

      Embryo quality evaluation

      The results showed that the quality of the 931 embryos (early cleavage stage and blastocysts), including the double embryo transfers (326 embryos in the study group and 605 embryos in the control group), was significantly improved by culturing them on AECC vs conventional culture medium (46% and 36% usable embryos, respectively, P = 0.003) (Table 2). Multivariable logistic regression analysis, conducted on the 931 fertilized oocytes, showed that an embryo is four times more likely to be transferable, regardless of the embryo stage (data for blastocysts and early cleaved embryos combined), when co-cultured (OR [95% CI]: 3.794 [2.847, 5.057], P < 0.001). Additionally, the blastulation rate was twice as high in embryos from the AECC group versus those placed in conventional culture medium (OR [95% CI]: 2.185 [1.603, 2.980], P < 0.001).
      Table 2Embryo quality (primary outcome)
      Group
      AECCCMP-value
      Between-group differences assessed with the chi-squared test.
      Number of total embryosn = 326n = 605
      Total of good-quality embryos
      Embryo quality assessed in fertilized embryos at early cleavage or blastocyst stage; includes double embryo transfers.
      , n (%)
      150 (46)217 (36)0.003
      D3 embryos, n (%)11 (3.4)45 (7.4)<0.001
      D5 embryos, n (%)99 (30.4)150 (24.8)<0.001
      D6 embryos, n (%)40 (12.3)22 (3.6)<0.001
      Good-quality blastocysts, n (%) (D5 and D6)139 (42.6)172 (28.4)<0.001
      AECC = autologous endometrial co-culture group; CM = conventional medium group.
      a Between-group differences assessed with the chi-squared test.
      b Embryo quality assessed in fertilized embryos at early cleavage or blastocyst stage; includes double embryo transfers.
      D5 and D6 embryos were three times (OR [95% CI]: 2.983 [2.185, 4.071], P < 0.001) and eight times (OR [95% CI]: 8.199 [3.466, 19.396], P < 0.001) more likely to develop into usable blastocysts when cultured on AECC compared with conventional medium, respectively.

      Pregnancy and live birth outcomes

      Pregnancy and live birth outcomes were assessed in the population of transferred embryos (n = 162). Overall, rates of biochemical pregnancies, clinical pregnancies and live births were comparable between AECC and conventional medium groups (Table 3). When assessed by type of embryo transferred, in the vitrified-warmed embryo transfer subgroup (n = 80), apparently higher rates of biochemical pregnancy (AECC = 58.5% vs conventional medium = 46.2%), clinical pregnancy (AECC = 46.3% vs conventional medium = 30.8%) and live births (AECC = 34.1% vs conventional medium = 25.6%) were found following AECC compared with conventional medium. Conversely, in the fresh embryo transfer (n = 82), apparently higher pregnancy rates (biochemical: AECC = 60.0% vs conventional medium = 66.7%; clinical: AECC = 45.0% vs conventional medium = 57.1%) and live birth rates (AECC = 42.5% vs conventional medium = 54.8%) were found following conventional medium compared with AECC (Table 3). Although none of the differences in rates in pregnancy and live birth rates across type of embryo transfer were statistically significant, the magnitude of the difference observed is nonetheless of clinical importance.
      Table 3Pregnancy and live birth outcomes following fresh versus vitrified-warmed embryo transfers (secondary endpoints)
      Group
      AECC
      Pregnancy outcomes assessed in transferred embryos.
      CM
      Pregnancy outcomes assessed in transferred embryos.
      Embryo transfers
       Fresh transfers, n4042
       Frozen embryo transfers, n4139
       All transfers, n8181
      Biochemical pregnancies
       Fresh transfers, n (%)24 (60.0)28 (66.7)
       Frozen embryo transfers, n (%)24 (58.5)18 (46.2)
       All transfers, n (%)48 (59.3)46 (56.8)
      Clinical pregnancies
       Fresh transfers, n (%)18 (45.0)24 (57.1)
       Frozen embryo transfers, n (%)19 (46.3)12 (30.8)
       All transfers, n (%)37 (45.7)36 (44.4)
      Single live births
       Fresh transfers, n (%)17 (42.5)23 (54.8)
       Frozen embryo transfers, n (%)14 (34.1)10 (25.6)
       All transfers, n (%)31 (38.3)33 (40.7)
      No significant between-group differences were identified with the chi-squared statistic.
      AECC = autologous endometrial co-culture group; CM = conventional medium group.
      a Pregnancy outcomes assessed in transferred embryos.

      Miscarriage rate

      No difference between the two groups has been observed for the miscarriages that happened during the study.

      Predictive model

      A predictive model was generated for the embryos that were still vitrified at the time of the analysis in order to estimate the pregnancy outcomes that would have been obtained with higher statistical power. The statistical coefficients obtained on the 150 vitrified embryos remaining were applied, respecting their randomization group. Cumulative probabilities showed a significant improvement in clinical pregnancy rates (57.03% in the AECC group vs 45.40% in the conventional medium group, P = 0.04) in favour of the AECC co-cultured embryos.

      Discussion

      The primary objective of this monocentric interventional study was to assess whether the use of AECC would improve embryo development and provide more usable blastocysts for all patients undergoing IVF/ICSI, independent of their history of infertility.
      Because the two populations in this study did not present any differences in terms of demographics or oocyte characteristics, it was possible to demonstrate that AECC significantly improved by 50% the number of quality blastocysts compared with the use of conventional culture medium. Thus, the AECC appears to enhance embryo development to blastocyst stage. These results were confirmed by the multivariate logistic regression analysis because the blastulation rate obtained with the autologous co-culture was twice as high as with the conventional medium. Indeed, D5 and D6 blastocysts co-cultured have three and eight times more chance, respectively, of being suitable for transfer, based on their quality criteria. A limitation of the design of this study is that the analysis was performed using embryos rather than patients. Because specific patient characteristics such as ovarian reserve and oocyte quality will impact on the overall results, this should be taken into consideration when assessing the findings. In addition, because the study was conducted within a private clinical setting and without access to a national registry, a follow-up of children born as a result of these treatments was not conducted. However, as with the introduction of any technology, it is an important step that should be carried out.
      The benefits of AECC in in-vitro treatments have been evaluated in several controlled studies. Improvements in terms of the number of blastomeres (
      • Barmat L.
      • Liu H.
      • Spandorfer S.
      • Kowalik A.
      • Mele C.
      • Xu K.
      • Veeck L.
      • Damario M.
      • Rosenwaks Z.
      Autologous endometrial co-culture in patients with repeated failures of implantation afterin vitro fertilization- embryo transfer.
      ,
      • Eyheremendy V.
      • Raffo F.G.E.
      • Papayannis M.
      • Barnes J.
      • Granados C.
      • Blaquier J.
      Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure.
      ,
      • Liu H.C.
      • He Z.Y.
      • Mele C.A.
      • Veeck L.L.
      • Davis O.
      • Rosenwaks Z.
      Human endometrial stromal cells improve embryo quality by enhancing the expression of insulin-like growth factors and their receptors in cocultured human preimplantation embryos.
      ,
      • Simón C.
      • Mercader A.
      • Garcia-Velasco J.
      • Nikas G.
      • Moreno C.
      • Remohí J.
      • Pellicer A.
      Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure.
      ) and a decrease in fragmentation (
      • Spandorfer S.D.
      • Barmat L.I.
      • Navarro J.
      • Liu H.C.
      • Veeck L.
      • Rosenwaks Z.
      Importance of the biospy date in autologous endometrial cocultures for patients with multiple implantation failures.
      ) leading to higher pregnancy rates were in favour of the co-culture.
      • Eyheremendy V.
      • Raffo F.G.E.
      • Papayannis M.
      • Barnes J.
      • Granados C.
      • Blaquier J.
      Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure.
      also showed the benefits of AECC on embryo development and clinical pregnancies in patients with repeated implantation failures. Furthermore, one trial had reported pre-embryo development improvement in patients with history of poor-quality embryos (
      • Spandorfer S.
      • Barmat L.
      • Navarro J.
      • Burmeister L.
      • Veeck L.
      • Clarke R.
      • Liu H.
      • Rosenwaks Z.
      Autologous endometrial coculture in patients with a previous history of poor quality embryos.
      ). AECC secretome analysis showed an enhanced expression of several growth factors that correlated with implanted blastocysts (
      • Dominguez F.
      • Gadea B.
      • Mercader A.
      • Esteban F.J.
      • Pellicer A.
      • Simón C.
      Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system.
      ,
      • Liu H.C.
      • He Z.Y.
      • Mele C.A.
      • Veeck L.L.
      • Davis O.
      • Rosenwaks Z.
      Human endometrial stromal cells improve embryo quality by enhancing the expression of insulin-like growth factors and their receptors in cocultured human preimplantation embryos.
      ,
      • Spandorfer S.D.
      • Barmat L.I.
      • Liu H.C.
      • Mele C.
      • Veeck L.
      • Rosenwaks Z.
      Granulocyte macrophage-colony stimulating factor production by autologous endometrial co-culture is associated with outcome forin vitro fertilization patients with a history of multiple implantation failures.
      ). A meta-analysis also established that the use of co-culture (human cells and other species combined) improved embryo morphology and implantation rate as well as clinical and ongoing pregnancy rates (
      • Kattal N.
      • Cohen J.
      • Barmat L.I.
      Role of coculture in humanin vitro fertilization: a meta-analysis.
      ).
      Although it was not possible to show a significant difference between the two groups in terms of pregnancy and live birth outcomes, it is clinically relevant to note that higher pregnancy and live birth rates were observed with the co-culture in case of frozen embryo transfer (FET) and higher rates with the conventional medium in the case of fresh embryo transfers (Table 3).
      However, based on the predictive model, according to the cumulative probabilities extracted from the embryos that were still vitrified at the time of the analysis, it was possible to achieve statistical power. Significantly, a 26% increase in clinical pregnancy rate and a 29% increase in live birth rate could be hypothetically predicted with the co-culture compared with the conventional medium. These results are together very interesting because they were corroborated by
      • Ohl J.
      • De Mouzon J.
      • Nicollet B.
      • Benoussaidh A.
      • Gouze J.
      • Viville S.
      • Menezo Y.
      Increased pregnancy rate using standardized coculture on autologous endometrial cells and single blastocyst transfer: A multicentre randomized controlled trial.
      . In this study, it was shown that AECC significantly (P < 0.05) increased the pregnancy rate per blastocyst transfer by 16% compared with the transfer of D3 embryos cultured on a conventional culture medium (
      • Ohl J.
      • De Mouzon J.
      • Nicollet B.
      • Benoussaidh A.
      • Gouze J.
      • Viville S.
      • Menezo Y.
      Increased pregnancy rate using standardized coculture on autologous endometrial cells and single blastocyst transfer: A multicentre randomized controlled trial.
      ). The present work allowed confirmation of the benefits of AECC on blastocyst development and more specifically in the context of vitrified embryos. Indeed, freeze-all cycles gained in popularity particularly because some authors demonstrated the advantages associated with FET when compared with fresh transfers, such as a reduction in the pre-term delivery (
      • Maheshwari A.
      • Pandey S.
      • Shetty A.
      • Hamilton M.
      • Bhattacharya S.
      Obstetric and perinatal outcomes in singleton pregnancies resulting from the transfer of frozen thawed versus fresh embryos generated throughin vitro fertilization treatment: A systematic review and meta-analysis.
      ) and a decrease in low birth weight and perinatal mortality (
      • Marianowski P.
      • Dabrowski FA.
      • Zygula A.
      • Wielgos M.
      • Szymusik I.
      Do We Pay Enough Attention to Culture Conditions in Context of Perinatal Outcome afterIn vitro Fertilization ? Up-to-Date Literature Review.
      ). Additionally, freeze-all cycles avoid the detrimental effects of ovarian stimulation that have been observed (
      • De Carvalho B.R.
      • Barbosa M.W.P.
      • Bonesi H.
      • Gomes Sobrinho D.B.
      • Cabral I.O.
      • Paes Barbosa A.C.
      • Silva A.A.
      • Iglesias J.R.
      • Nakagawa H.M.
      Embryo stage of development is not decisive for reproductive outcomes in frozen-thawed embryo transfer cycles.
      ,
      • Fatemi H.
      • Popovic-Todorovic B.
      Implantation in assisted reproduction: A look at endometrial receptivity.
      ,
      • Gardner D.K.
      • Schoolcraft W.B.
      • Wagley L.
      • Schlenker T.
      • Stevens J.
      • Hesla J.
      A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization.
      ,
      • Roque M.
      • Valle M.
      • Guimarães F.
      • Sampaio M.
      • Geber S.
      Freeze-all policy: Fresh versus frozen-thawed embryo transfer.
      ) and combined with the results from endometrial receptivity tests (
      • Haouzi D.
      • Dechaud H.
      • Assou S.
      • Monzo C.
      • De Vos J.
      • Hamamah S.
      Transcriptome analysis reveals dialogues between human trophectoderm and endometrial cells during the implantation period.
      ,
      • Simón C.
      • Mercader A.
      • Garcia-Velasco J.
      • Nikas G.
      • Moreno C.
      • Remohí J.
      • Pellicer A.
      Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure.
      ) allow the transfer of quality blastocysts at a more propitious time.
      In addition, it has been demonstrated that lower oxygen concentrations benefit blastocyst development (
      • Bontekoe S.
      • Mantikou E.
      • van Wely M.
      • Seshadri S.
      • Repping S.
      • Mastenbroek S.
      Low oxygen concentrations for embryo culture in assisted reproductive technologies.
      ,
      • Meseguer M.
      • Pellicer A.
      The most well kept secret, embryo culture media : a smart reveal from an expert.
      ) and this represents a difference in these study groups, however the co-culture group did not benefit from lower O2 concentrations and it could be projected that this would have only increased the efficiency of the AECC.
      As the literature has shown, blastocysts are more likely to lead to ongoing pregnancies and live births (
      • Gardner D.K.
      • Lane M.
      Alleviation of the ‘2-cell block’ and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters.
      ,
      • Papanikolaou E.
      • Kolibianakis E.
      • Tournaye H.
      • Venetis C.
      • Fatemi H.
      • Tarlatzis B.
      • Devroey P.
      Live birth rates after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and meta-analysis.
      ,
      • Schwärzler P.
      • Zech H.
      • Auer M.
      • Pfau K.
      • Gobel G.
      • Vanderzwalmen P.
      • Zech N.
      Pregnancy outcome after blastocyst transfer as compared with early cleavage stage embryo transfer.
      ,
      • Simón C.
      • Mercader A.
      • Garcia-Velasco J.
      • Nikas G.
      • Moreno C.
      • Remohí J.
      • Pellicer A.
      Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure.
      ). The rate of quality blastocysts obtained is thus an important factor for ART, consequently contributing to elective single embryo transfer (eSET) success combined with the use of vitrification and preimplantation genetic testing (
      Practice Committee of the American Society for Reproductive Medicine and Practice Committee of the Society for Assisted Reproductive Technology
      Blastocyst culture and transfer in clinically assisted reproduction: a committee opinion.
      ).
      Data from this study suggest that the use of co-culture exploiting the patient's own endometrial cells could favour the development of embryos into good-quality blastocysts, and may be specifically beneficial to freeze-all cycles. While several teams have concentrated their works more specifically on patients with IVF failure (
      • Eyheremendy V.
      • Raffo F.G.E.
      • Papayannis M.
      • Barnes J.
      • Granados C.
      • Blaquier J.
      Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure.
      ,
      • Spandorfer S.
      • Barmat L.
      • Navarro J.
      • Burmeister L.
      • Veeck L.
      • Clarke R.
      • Liu H.
      • Rosenwaks Z.
      Autologous endometrial coculture in patients with a previous history of poor quality embryos.
      ,
      • Spandorfer S.
      • Pascal P.
      • Parks J.
      • Clark R.
      • Veeck L.
      • Davis O.
      • Rosenwaks Z.
      Autologous endometrial coculture in patients with IVF failure: outcome of the first 1,030 cases.
      ), data from this study suggest that all patients could benefit from AECC. However, a confirmatory study using patients as the denominator rather than embryos would help to confirm this proposal, as well as a follow-up of children born as a result of these treatments.

      Acknowledgements

      This work was supported by Laboratoires Ovo Inc. (Ovo Labo). We thank Ovo R&D, who provided logistical assistance, data collection, data analysis and regulatory follow-up of this clinical study. We especially thank the embryologists from Ovo Fertility for logistical and technical assistance and data acquisition.

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      Biography

      Dr. Kadoch is an associate professor at the Faculty of Medicine at the University of Montreal. He is currently Head of the Medicine and Reproductive Biology department at the Centre Hospitalier de l'Université de Montréal (CHUM). He is one of the co-founders of the ovo clinic where he serves as medical director of ovo labo and ovo r&d. Dr Kadoch has completed his medical studies at the Faculty of Medicine at the University of Pierre and Marie Curie in Paris. Then, he completed a fellowship in reproductive endocrinology and infertility at the University of Montreal. He was the director of the university program of Gynecologic Reproductive Endocrinology and Infertility (GREI) and he is a committee member for examinations for the Royal College of Physicians and Surgeons of Canada in GREI.
      Key message
      Autologous endometrial co-culture (AECC) increases the development of good-quality blastocysts compared to conventional culture. This is the first study demonstrating the benefits of AECC in couples undergoing IVF/ICSI with different causes of infertility.