Session 5 Q&A

 

 

Questions for David:

     Evelyn Llerena Cari: How long does folliculogenesis take since the initial recruitment of primordial follicles?

     This was address during the session

     Evelyn Llerena Cari: During the primordial follicles quiescence stage, do you think there is still communication between the flattened pre-granulosa cells and the oocyte?

     This was addressed during the session

     Charlene Alouf: Since the cohort destined for natural or IVF stimulated recruitment is determined months in advance do you think that building a healthier ooycte(s) should involve a pretreatment 2-3 months prior to the cycle - at the multilaminate follicle?  Do you think that this perhaps occurs in the rebound pregnancy that sometimes happens after a failed IVF cycle -especially in couples with a considerable history of infertility.

     Excellent point and that we often observe the rebound effect after a failed cycle has lead many of us to think that it is the endogenous natural gonadotropins that rebound and release early antral follicles that were not responding to stim cycle treatment. In theory, yes we should be considering at least lifestyle and diet and/or exposure to environmental hazards as all possibly impacting this critical early stage of oocyte development-hence alerting patients is a good thing. But do we know what might directly positively influence oocyte quality at time of retrieval or ovulation? At this time the answer is no.

     Evelyn Llerena Cari: How successful (and “efficient”) is IVM in human primordial follicles?

     So far as Dr Telfer mentioned, maybe 30% of what makes a retrievable COC will mature but so far, we can see that developmental competence is incomplete in these oocytes. Advances in IVM for human oocytes may allow us to revisit conditions that could improve quality work that is underway. Bear in mind, that of all the primordial follicles in an step one preparation, very few get to an antral follicle stage. So as in vivo, attrition of follicles is a major challenge in this field.

 

 

Questions for Evelyn:

      Jayaprakash Divakaran: what is the time duration to develop primordial germ cells to Mature oocytes in vitro

     In our system it takes 19-21 days to grow from Primordial Follicles to obtain oocytes large enough to place in IVM media

     Evelyn Llerena Cari: Do you select the human primordial follicles based on histological features (flattened granulosa cells around the oocyte)?

     We do not select Primordial Follicles. The ovarian Cortex containing the Primordial follicles is prepared for step one of the culture system. Isolated Primordial Follicles will not activate and grow!

     Evelyn Llerena Cari: Were you disrupting the HPO axis?

     All of this work is in Vitro

     Teri Ord: Have you looked at cortical granules in the matured oocytes?

     Not in any detail at this stage

     pentti koivisto: She mentioned a membrane? What kind of membranes

     track-etched nucleopore membranes (Millipore)

        M. McLaughlin , D.F. Albertini , W.H.B.Wallace , R.A.Anderson E.E. Telfer.(2018) Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system. Molecular Human Reproduction 24:135-142

     asmaa sanjary: who can induce final maturation to mll oocyte?

     See publication in references:

     M. McLaughlin , D.F. Albertini , W.H.B.Wallace , R.A.Anderson E.E. Telfer.(2018) Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system. Molecular Human Reproduction 24:135-142

     Shaun Rogers: Have assessments been made of the chromosomal makeup and ploidy rate of the extruded first polar body or the oocyte by NGS? I understand the limitations of this.

     Not yet but we hope to do this

     Sule Dogan: Have you done any IVG experiments in older patients especially women over 40 years old?

      Our ethical approval currently only allows patients under 40 to donate tissue

     celal kaloglu: what is the 02 level to improve PB size?

     5:5: 90

     Aysin Bak: What is your opinion about Kyle orwig studies. They use primates

     Is this male studies that you are referring to? I am not sure what is being asked here.

     Nuno Costa Borges: Wonderful presentation Evelyn. Have you had the chance to look at the chromosome status of the human oocytes produced and the complementary polar bodies?

     Thank you Nuno, we need to do this.

     Pablo Hurtado: for Evelyn - Regarding the samples from transgender patients. Are these ovaries exposed to hormones from the person’s treatment before being obtained? Have you seen a difference between treated vs non treated?

     Yes we do particularly in the primordial population

     I ask to what extent your research goes on for example cleavage , blastulation and so on or if it stopped at the pn stage.

     No fertilization yet as we require a license for this

     Laura Ball from Picton Lab in Leeds: I totally understand the desire to reduce the time follicles spend in culture. Is there any possibility that folliculogenesis could be completed in less than the 19-21 days you report, without detrimental impacts?

     I don’t know!

     H Fung: Many studies indicated in vitro maturation has high spindle errors, so PGAT of Oocytes should be very useful.

     Agreed and we now have a Wellcome Trust Funded Project to  look at this in more detail

     Jessica Hernández Contreras: In humans, using this method can reduce fertility problems associated with the oocyte?

     Potentially

     Salman Aldakhilallah: When it comes to classification of follicles and oocytes, there is no single international accepted guidelines for classification of such? Why is that?

     We use Peters and Pedersen’s classification. The issue with the systems is that many have been designed for rodent follicles and there are some differences in humans. However, I think all systems agree on basic stages of development. Our papers outline stages.

     Can in vitro growth of oocytes provide better quality oocytes for older patients?

     Potentially but a lot more work is needed.

     Is quality determined by the age of the oocyte or the environment that it develops in?

     Both. Age clearly has an effect but if tissue was stored at a young age we would be working with young oocytes.

 

 

 

Practical technique questions for Evelyn

     Mariav: which type of needles do you use to isolate the follicles from the tissue? as they are really tiny.

     Primordial follicles are not isolated but once they are growing they are isolated from the cultured ovarian cortex.

     During Step 1 growing follicles were observed on the surface of the cortical fragments and within 8 days of culture visible follicles of 100–150 μm in diameter were observed. These growing follicles were mechanically dissected with 25 gauge needles and those with an intact basement membrane were selected for further culture.

     Jayaprakash Divakaran: Instead of culturing the excised ovarian tissue Is it advisable to extract the primordial follicles and isolate by cell sorter and culture it in vitro?

     No, Isolated Primordial follicles do not activate growth in vitro!

     Emma Park: Do you reccomend denuding cumulus-oocyte complexes to ensure only 1-2 layers of cumulus cells before placing them on the membrane?

     No

     ZEKI BEYHAN: Why not keep the COC in the follicle until completion of maturation as in natural case?

     To avoid having to maintain large antral follicles in vitro and to focus on the oocyte and its surrounding cells.

     Lada Diachenko: how do you extract COC from in vitro grown follicles?

     Individual follicles were monitored and at the end of 8 days in Step 2, follicles with antral cavities were observed. COCs were retrieved from intact healthy cultured follicles by gentle pressure. The aim of this stage was to obtain oocytes that were at least a 100 μm in diameter for IVM

 

 

 

 

 

Some Relevant Recent Publications:

        Maidarti M, Clarkson YL, McLaughlin M, Anderson RA,Telfer EE. Inhibition of PTEN activates bovine non-growing follicles in vitro but increases DNA damage and reduces DNA repair response. Hum Reprod 2019;34: 297-307.

        Telfer E.E. (2019) Future developments: In vitro growth (IVG) of human ovarian follicles. Acta Obstet Gynecol Scand. doi: 10.1111/aogs.13592

        Telfer EE, Anderson RA (2019) The existence and potential of germline stem cells in the adult mammalian ovary. Climacteric.  22(1):22-26.

        M. McLaughlin , D.F. Albertini , W.H.B.Wallace , R.A.Anderson E.E. Telfer.(2018) Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system. Molecular Human Reproduction 24:135-142

        Clarkson YL, McLaughlin M, Waterfall M, Dunlop CE, Skehel PA, Anderson RA, Telfer EE. (2018) Initial characterisation of adult human ovarian cell populations isolated by DDX4 expression and aldehyde dehydrogenase activity. Sci Rep.8:6953

        Anderson RA, Telfer EE (2018) Being a good egg in the 21st century. Br Med Bull: 127(1):83-89.

        Anderson RA, Wallace W Hamish B, and Telfer EE (2017) Ovarian tissue cryopreservation for fertility preservation: clinical and research perspectives. Human Reproduction Open 1:

        McLaughlin M, Kelsey TW, Wallace WH, Anderson RA, Telfer EE (2017) Non-growing follicle density is increased following adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) chemotherapy in the adult human ovary. Hum Reprod.32:165-174

        Albertini DF, Telfer EE (2016) Deconstructing the winding path to the recapitulation of mammalian oogenesis ex vivo.Proc Natl Acad Sci U S A. 113:9956-7.

        Anderson RA, McLaughlin M, Wallace WH, Albertini DF, Telfer EE (2014) The immature human ovary shows loss of abnormal follicles and increasing follicle developmental competence through childhood and adolescence. Human Reproduction 29: 97-106

        McLaughlin M, Kinnell H, Anderson RA, Telfer EE (2014) Inhibition of phosphatase and tensin homolog (PTEN) in human ovary in vitro results in increased activation of primordial follicles but compromises development of growing follicles Molecular Human Reproduction 20: 736-44