Focus on Reproduction

Here’s a brief overview of the novelties in embryology presented in during this annual meeting of ESHRE.

Recent findings on ‘Oliana strings’, named after the embryologist who characterised them, were discussed on Monday. Such structures, observed in 77% of embryos, are in fact an extension of the corona cell interacting with the oolemma. They are associated with embryo fragmentation but have no effect on ploidy or implantation rate.

Next, the highly controversial question of assisted hatching was addressed by Kenji Ezoe, who provided new insights into the molecular mechanisms (increased expression of integrin α5β1 mRNA in fully hatched embryos) involved in the attachment of a fully hatched embryo after warming. He concluded that blastocyst adhesion rate was 20% and 70% higher in complete ZP removal group as compared to the intact and partially hatched embryos.

Culture media are still the subject of great attention. Ellen Casser described the creation monozygotic twin mouse embryos and their culture in different culture media to test their impact on the developing embryos and subsequent pups. It also seems that the composition and physico-chemical properties of culture media vary throughout storage; we were warned that commercial culture media may be contaminated with company staff cell-free DNA! The question of any potential impact on embryo remains unanswered…

Non-invasive approaches to select the most viable embryo for a singleton pregnancy are still being developed: Dr Sakkas proposed non-invasive fluorescence lifetime imaging microscopy (FLIM) as a system for comprehensive embryo metabolism measurement. It was also shown that culture medium lipid fingerprint and the microRNA (MiR-142-3p) are potential markers for implantation success. Use of time-lapse systems was also behind the identification of novel phenomena associated with fertilisation known as cytoplasmic waves, which may predict embryo implantation.

Debbie Montjean, SIG Embryology Junior Deputy

Single-cell omics, namely single-cell sequencing, was selected as the method of the year in 2013 by Nature Publishing Group (Nawy, 2014). A few years later, a comprehensive transcriptional map of 1529 individual cells from 88 human preimplantation embryos using single-cell RNA-sequencing (RNA-seq) was published (Petropoulos et al, 2016). Here in Geneva Dr Jeroen Krijgsveld presented the first study of single-cell proteome analysis in human oocytes. Single-cell proteome analysis reflects the full content of proteins within the cell, the key determinants of cellular function, and therefore has the best potential for predicting/demonstrating oocyte competence.

Nevertheless, any analysis of the full proteome is a challenging task  – the proteome is large and of unknown complexity, and even more on the single cell level because of the limited sample input for mass spectrometry analysis.

It is wonderful to see that Dr Krijgsveld’s group has overcome these limitations by presenting a novel strategy to extract, purify and digest proteins with a novel magnetic bead-based procedure, thereby minimising sample losses and increasing overall sensitivity of detection. As a result, they are now able to routinely detect, identify and quantify  around 600 proteins from a single human oocyte. Their comprehensive single-oocyte analysis demonstrates that the proteome composition distinguishes immature oocytes from mature MII oocytes. Furthermore, they showed that oocytes that are matured in vitro in the presence of FSH and HCG and in a co-culture with cumulus cells are more similar to in vivo-matured cells than the cells matured with FSH and HCG only, thus indicating the importance of the ovarian niche during the maturation process. They also demonstrated that, based on the proteome data, co-culture with cumulus cells can be efficiently replaced by addition of the protein vimentin (which is an important component of mesenchymal cells, including granulosa cells).

Altogether, these results indicate that technological advances now enable proteome analysis at the single oocyte level, providing a powerful tool for optimising and improving oocyte maturation procedures.

Signe Altmäe, Junior Deputy SIG Reproductive Genetics

References

Nawy T. Single-cell sequencing. Nat Methods 2014; 11: 18.

Petropoulos S, Edsgärd D, Reinius B, et al. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos. Cell 2016; 165, 1012–26. doi:10.1016/j.cell.2016.03.023

The European IVF Monitoring (EIM) Consortium project began almost 20 years ago, with full support from the ESHRE. It was initiated by those who clearly understood that reliable information about the results of ART is absolutely necessary for clinicians, scientists, patients, politicians and wider society. From its inception, EIM has consisted of a group of national representatives who volunteered to collect ART data in their own countries (at present, half of the participating countries have official national registries).

The 2014 report presented during this Annual Meeting is a preliminary one since some countries have not provided their data yet (UK being the largest). Nevertheless, comparing the available data with those in the 2013 final report (which has now been submitted for publication) we see an increase in total numbers of treatment cycles (3%), mainly because Spain and Russia greatly increased their registry requirements. According to the data we received, Spain was the most active European country using ART, followed by Russia and France. Because of their particularly wide use in Spain, frozen embryo replacement and egg donation cycles also increased significantly. The recently seen proportion of 2:1 in the number of ICSI vs IVF cycles was once again evident in 2014..

Effectiveness in terms of clinical pregnancy per ovarian aspiration was shown to be quite stable compared with 2014, while the rate of clinical pregnancies per embryo transfer was slightly higher in all techniques except IUI (a treatment not considered ART in most countries).

Reported complications were once again very low, but we cannot exclude that this results from some underreporting.

Although transfers of three or more embryos still represent more than 40% of total transfers in a few countries, globally the proportion of single embryo transfers continues to increase in Europe (to almost 35% in 2014). The proportion of multiple deliveries showed a small decline to around 18% in twins (with triplets still at 0.5%).

Carlos Calhaz-Jorge, Chairman EIM Steering committee

Human Reproduction Keynote lecture by Carlos Simon

For this honorary lecture again the most frequently downloaded original Human Reproduction paper of the preceding year was selected by the editors of the journal. For the first time this year also the paper’s AltMetrics score was taken into account, as a reflection of social media attention.

In a full auditorium (picture; 3499 seats taken, one left), Dr Carlos Simon presented the Human Reproduction Keynote lecture 2017. In this pilot study, the authors asked themselves whether bone marrow-derived stem cells could offer a safe and efficient therapeutic approach for patients with refractory Asherman’s syndrome, and a wish to conceive. In a group of ‘difficult’ patients resistant to conventional treatment they found that in the first 3 months, autologous cell therapy in conjunction with hormonal replacement therapy increased the volume and duration of menses as well as the thickness and angiogenesis processes of the endometrium while decreasing intrauterine adhesion scores. 11 Asherman patients completed the study. All exhibited an improved uterine cavity 2 months after stem cell therapy. Endometrial thickness increased from an average of 4 mm to 7 mm. The cell therapy increased the mature vessel density and the duration and intensity of menses in the first 3 months, with a return to the initial levels 6 months after the treatment. Three patients became pregnant spontaneously, resulting in one baby born, one ongoing pregnancy and one miscarriage. In addition, 7 pregnancies were recorded after 14 embryo transfers, resulting in 3 biochemical pregnancies, 1 miscarriage, 1 ectopic pregnancy, 1 baby born and 1 ongoing pregnancy. Observational studies like this one are the first step towards more rigorous, comparative studies. Dr Simon pointed out that limitations of this pilot study include the small sample size and the lack of controls, but he suggested that autologous bone marrow-derived stem cell therapy is a promising therapeutic option for patients with otherwise incurable endometrial pathologies and a desire to conceive. He finally mentioned the collaboration between the authors and the expert reviewers and editors of Human Reproduction. He applauded it, thanked the journal and characterized the review process as a time consuming but very rewarding experience.

 Hans Evers, Editor in Chief of Human Reproduction

After a decade in planning and delivery and at a cost of surely a few million euros, the results of the ESTEEM trial of aneuploidy testing with polar body analysis by array CGH were finally delivered today. Karen Sermon, co-ordinator of the trial following the retirement of its instigator Joep Geraedts, said that at the time of planning this approach made sense – polar body analysis would avoid the problem of mosaicism in the embryo, and most live births with chromosomal problems are anyway maternal in origin. A pilot study was launched to test feasibility and the RCT – ESTEEM – would eventually follow.

And so today, four years after recruitment began we had the initial results. The trial’s first primary outcome question was whether full chromosome analysis of both polar bodies would increase the likelihood of a live birth within one year – and the answer to that question is unequivocally ‘no’. Among the 205 patients allocated to aneuploidy testing there were 41 with at least one live birth; and among the 191 randomised to no intervention there were 42. A score draw.

It would be easy to describe the results as disappointing, but unlike history’s previous landmark PGS trial (from Amsterdam with FISH) delivery rates now were not adversely affected by screening. It was also clear from the smaller print details that that there were far fewer transfers in the screening group (178 vs 270), suggesting that aneuploidy testing did at least make for more efficient treatment. The bare details of the data – without additional calculation – also made a suggestion that the miscarriage rate in the PB group was substantially lower than in the no intervention group. These were important positive findings for trial participant Cristina Magli, but would they be enough in the patient’s view to compensate for no benefit in delivery rate?

Of course, other questions remain, and opinion was divided among those in the room listening to the results. There was debate on both their strict interpretation and on their wider implications. For example, can we consider this study a model for the whole broader concept of aneuploidy testing? Is it ever possible for screening to really increase live birth rate? Clearly, there’s much work yet to be done in the calculations and conclusions of ESTEEM, but there’s no doubt that the final published paper will attract a fair amount of comment. And no-one present today was in any doubt that this was a monumental work of diligence, determination and scientific discipline. So still in high esteem.

* The trial was funded by ESHRE and by Illumina, who freely provided the arrays for the CGH.

Simon Brown, Editor of Focus on Reproduction

It’s Sunday evening. You stand outside the huge building bedecked with ESHRE flags. You pause, take a deep breath, and enter ‘Conference World’. Signs everywhere. An App to get to grips with. And thousands of people. Thousands. For three days.

The ESHRE Meeting has its own unique dynamic and atmosphere. Despite moving around European cities, there is a reassuringly steady rhythm to the Annual Meeting. As we move through the established initial pattern of events; walking in for the first time, waving to friends as we queue for our badge, watching the ‘seniors’ conspicuously greet each other at the front of the hall at the Opening Ceremony, and looking for friends and colleagues at the opening reception, we all become part of it. Part of the biggest conference on reproductive medicine and science in the world.

As we move though from the palpable spring-like energy on Monday, through the high summer of Tuesday, to the autumnal melancholy of the meeting drawing to its Wednesday close, we share a journey. If you just come to listen to the science and meet old and new friends, you will go home tired, probably wiser and hopefully inspired. But decide to become an active member of the society, and the Annual Meeting becomes so much more.

In recent years, the structure of ESHRE has really opened up. Everyone with an interest, energy and wish to contribute can do so. There are many ways, but the core of the society lies in its Special Interest Groups. Find one that addresses your interest. Go to its business meeting. Take the microphone and give your view. Propose a topic for a Campus, offer your candidacy for the coordinating committees. Do so, and a new world opens of friends, colleagues and a real chance to shape the future of your field and your society.

So this year, even if it is your first ESHRE meeting why not do it? Engage.

And who knows, one day you too will be conspicuously greeting your friends in the front row at the Opening Ceremony!

Nick Macklon, Member of the Executive Committee

Monday was IUI day. After an opening keynote session which saw standing room only in a hall with 3000 seats, there was also a full house for an early free communication session on IUI in unexplained infertility. Both the opening two reports of the session made a strong case for IUI itself, and in contradiction to the recommendations of NICE, which, on the basis of limited evidence, had recommended in 2013 two years of wait-and-see to be followed by IVF. Indeed, said Cindy Farquhar, this controversial recommendation was the very basis of her New Zealand trial, which compared three cycles of IUI (with clomiphene citrate) with three cycles of expectant management on cumulative LBRs. Dr Farquhar, from the University of Auckland, said that the NICE recommendations were based on just two trials in unexplained infertility, one of which included IUI without stimulation. Results of the study, which had randomised 201 patients with unexplained infertility to IUI or expectant management, showed that the former was associated with a three-fold greater live birth rate than the latter (31% vs 9%). ‘IUI with clomiphene,’ said Dr Farquhar with a little understatement, ‘may be offered to couples with unexplained infertility as a safe and effective treatment.’

This too was the conclusion reached by another RCT, this from the Netherlands, whose presenter, Dr Monique Mochtar from the Amsterdam Medical Centre, even went so far as to recommend IUI with clomiphene stimulation as first choice in unexplained infertility – less invasive, less expensive and just as effective as FSH.

The study, performed in 24 fertility centres in the Netherlands, randomised 369 women to IUI with FSH and 369 women to IUI with clomiphene. Results showed that 31% (113 women) had an ongoing pregnancy following IUI-FSH and 26% (97 women) had an ongoing pregnancy following IUI-CC. Results also showed that five women (1%) had a multiple pregnancy following IUI-FSH and eight (2%) had a multiple pregnancy following IUI-CC – again, a statistically non-significant difference.

In an interview with me after her presentation Dr Mochtar suggested that the NICE guidelines might well need revision following publication of these two trials. She had no doubt that in her recommendations treatment should start with IUI stimulated with clomiphene. However, it should be noted that 48 of the 210 ongoing pregnancies (23%) in the Dutch study were achieved by natural conception. This too is in line with the natural conception rate reported from other trials, thus suggesting that ‘expectant management’ might not be such a hopeless alternative. As one UK IVF veteran said, couples are usually better off just ‘getting on with it’.

Simon Brown, Editor of Focus on Reproduction