New Markers of Ovarian Response

In the last years, much progress has been made in the development of analytical and ultrasound markers to evaluate the ovarian reserve and therefore be able to predict more accurately the ovarian response and design specific protocols that improve the efficacy and safety of IVF treatments.

However, our knowledge about ovarian physiology is still unsatisfactory. We need to find new markers of follicular development that give us information about oocyte quality and open a path for research to improve it in the future. The first paper is going to tell us about the development of these new markers that participate in the processes of follicular maturation. It opens a very interesting path for the future.

In the second paper the concept of "ovarian sensitivity" will be developed, trying to reveal the reasons why women, with the same reserve, have different responses to ovarian stimulation. At the same time we will debate/discuss if that better response is related to the oocyte quality and therefore to the results both in the laboratory and in the clinic.

It is necessary to move forward in the knowledge of the ovarian physiology, not only in ovarian reserve terms, but also in oocyte quality in order to improve the prognosis of our patients.

What's the ideal stimulation?

What’s an ideal stimulation?

Ovarian stimulation (OS) constitutes an integral part of assisted reproductive techniques. Albeit the first successful in-vitro fertilization (IVF) attempt was conducted in a spontaneous menstrual cycle, soon thereafter OS was introduced to induce development of multiple dominant follicles, and to mature more oocytes to improve chances for conception. Clomiphene citrate, human menopausal gonadotropins and exogenous gonadotropins emerged as the "armamentarium" of drugs inducing OS.

Over the last 30 years, they have become the mainstay in fertility treatment worldwide. In this session of the third edition of Meeting the Experts, two prominent specialists in the field will provide insights on the different options for OS in IVF/ICSI in the search for that "perfect" protocol that will give us an optimal ovarian response, adequate number of oocytes resulting into competent embryos providing good live birth rates and also taking into account safety and patient compliance.

Individualization. Is it worth it?

It has always been important and a fundamental part of the doctor's role to take each patient's individual characteristics into account in medical interventions, to obtain the best results and minimise undesired effects. What is more, our profession is increasingly evolving towards personalised medicine. So, for example, in recent decades we have seen how we have adjusted anti-tumour therapies to the genetic and molecular make-up of our patients' tumours, and with the unstoppable advance of knowledge about genetics, in the next few years, we will be looking at designing specific and tailor-made treatments in many fields of medicine.

Reproductive medicine is no exception to this trend and individualised treatment is becoming an increasingly essential part of its success. However, so far, the methods available for personalising treatments are limited. This is why it is important to share knowledge and continue research work on developing new tools that enable us to offer our patients the best options. In this session, the current state of the subject will be described and there will be a discussion on future possibilities.

The trigger and the "freeze all strategy"

Conventionally, most IVF embryos are transferred in a fresh cycle. Any surplus embryos available are frozen and stored for later use if no pregnancy results from the fresh embryo-transfer or when patients wish to expand their family. The first live birth after transfer of a thawed cryopreserved embryo was reported back in the early '80s. Nonetheless, the use of frozen–thawed IVF embryos has consistently lagged behind fresh embryo transfer.

More recently, improvements in vitrification protocols have allowed elective freezing of all embryos, followed by transfer in a subsequent cycle (eFET), also known as the ‘freeze-all’ strategy. Initially, this strategy was indicated for hyper-responders, as these individuals are at a high risk of developing ovarian hyperstimulation syndrome (OHSS). Later, it was hypothesized that controlled ovarian stimulation could lead to negative effects in the endometrium and implantation. Hence, it was suggested that performing eFET would not only decrease the risk of OHSS, but also improve the reproductive outcomes in IVF cycles. In contrast, eFET may increase the cost of treatment and workload, requiring additional embryo manipulation and an increase in the time to live birth. In this session of the Meeting the Experts, Prof. Ben Willem Mol will appraise and consider the implications of adopting such a strategy in routine clinical practice.

For the "freeze-all" strategy a bolus of a gonadotrophin-releasing hormone (GnRH) agonist is usually employed as trigger agent. This approach was possible due to the introduction of the GnRH antagonist protocols for the prevention of a premature LH surge, as the GnRH agonist will displace the GnRH antagonist from the GnRH receptor and induce a flare-up of both endogenous LH and FSH, resembling the natural midcycle surge of gonadotrophins for final follicular maturation. Although the surge of gonadotrophins elicited by a bolus of GnRH agonist differs from the natural midcycle of gonadotrophins in duration and profile, it has previously been shown to effectively stimulate final oocyte maturation and ovulation. In the first part of the session Dr. Juan Carlos Castillo will expand on the potential benefits of the GnRH agonist as a trigger agent beyond OHSS prevention.

Endometrial Receptivity Assessment (clinical markers)

Endometrial receptivity is defined as the state, in which the endometrium allows embryo implantation. Endometrial biopsy is postulated as an invasive method to determine endometrial receptivity. It can be used to analyze both histological parameters and their molecular changes. The Ultrasound scan, with all its possible applications, such as the use of 3D ultrasound, three-dimensional vascularization or the measurement of uterine contractions, is presented as the non-invasive method of analysis of this receptivity.

Endometrial Receptivity Assessment (the molecular biology perspective)

Mitochondria constitute the powerhouse of cells, producing the energy required for all cellular functions, and playing a vital role in cell function. The mtDNA copy number is strictly regulated ensuring that mitochondria can generate appropriate levels of energy. Altered mtDNA copy number has been shown to be involved in cancer, neurodegeneration disorders, ageing and diabetes. Usually, mtDNA molecules of all the cells of an individual are homoplasmic, carrying only one genome with a given nucleotide sequence. However, recent studies have shown that mtDNA heteroplasmy is common in the general population. Recently, there has been an increasing research on mtDNA as biomarker of embryo implantation. Although reports showed that high levels of mtDNA in blastocyst are associated with low implantation potential, other publications were unable to confirm this. Confounding factors may influence the mtDNA copy number in euploid embryos and could be the reason of this discrepancy. In addition, it is unclear whether high levels of mtDNA are a constant feature in some group of women or whether it depends on each embryo, showing variability among sibling embryos from the same cohort. On the other hand, it has been speculated that both mtDNA heteroplasmy and copy number contribute to mitochondrial function. Moreover, the effects of mtDNA heteroplasmy and copy number were only discussed separately, thus remaining inconclusive. Next generation sequencing (NGS) offers the great sensitivity required for the detection of low levels of heteroplasmy. Thus, NGS has provided the opportunity to conduct in-depth analysis allowing the study of mtDNA heteroplasmy and copy number simultaneously.

In our group, we investigated what factors could condition mtDNA levels. We examined whether maternal and embryo factors influenced mtDNA quantity. Moreover, we investigated the influence of mtDNA copy number and heteroplasmy in euploid non mosaic embryos on IVF outcome. Lastly, we examined the variability in the levels of mtDNA among sibling euploid embryos from the same cohort.

From our results we can conclude that elevated mtDNA embryos have a lower chance to produce an ongoing pregnancy. MtDNA is higher in older women and it is dependent upon the cell divisions that preceded biopsy. Our data suggest that mitochondrial activity could be a balance between functional capacity and relative mtDNA copy number. Finally, we report that mtDNA content is not a marker of prognosis amongst patients because it is variable among the embryos from the same cohort, thus mtDNA could be used as an additional marker of embryo competence and a tool to prioritize the euploid embryo to be transferred.

Time to pregnancy

  • Session I
  • Saturday 28th September 2019
  • Jorge Ten

In this session, we will discuss one of the most important markers in assisted reproduction today, such as the "time to pregnancy". In a common goal, both patients, as well as clinicians and embryologists, seek to shorten the time of pregnancy, giving optimal safety and efficacy. From the point of view of the Gynecologist, our Medical Director, Dr. Rafael Bernabeu will explain the different approaches, which will allow us to obtain an appropriate number of competent gametes, and an optimal endometrium, both essential for the achievement of a pregnancy.

In addition, Dr. Bernabeu will discuss the different strategies, such as the accumulation of oocytes and/or embryos, which may be valid for certain reproductive disorders, such as low ovarian reserve. From the point of view of the Embryologist, Dr. Etienne Van den Abbeel will focus on new methods of selection and embryo culture, which will allow selecting the best embryo for transfer, with more possibilities of obtaining a single and ongoing pregnancy and subsequent birth of a healthy child.

PGT-A as a tool in the management of IVF patients

In this session on preimplantation genetic testing (PGT) we will be discussing two topics that will highlight the main debates and controversies in this field.

Embryo aneuploidy is one of the leading causes of failure in IVF cycles. Although selecting euploid embryos by means of PGT-A (Preimplantation Genetic Testing for Aneuploidy) has been put forward to improve success rates in IVF cycles, recent studies have questioned the benefits. In response to these criticisms, other authors recommend limiting PGT-A to certain cases, such as recurrent miscarriage and advanced maternal age. The issue is open to debate.

To carry out PGT-A, an embryo biopsy in D+3 or D+5 is required, which may reduce its ability to implant. New methods have been developed that propose using the embryo culture medium as a source of DNA. Such an alternative would avoid embryo biopsy but this new methodology has yet to confirm its diagnostic ability.

Looking inside the embryo

  • Session III
  • Saturday 28th September 2019
  • Jorge Ten

Recent studies suggests that oocyte mitochondrial content and function may be related to implantation success and embryo viability. Our research group, led by the Scientific Director, Dra. Belén Lledó, has recently published an article addressing the importance of mitochondria in reproduction. In this context, Dra. Lledó will comment on our own results obtained at Instituto Bernabeu, which will deal not only with the number of copies of mitochondrial DNA, but also with the functional capacity of the mitochondria and its role in reproduction.

The embryologist is the one ultimately responsible for the success of the IVF laboratory. Therefore, an optimal embryo selection is essential to improve clinical outcomes is an assisted reproduction program. In this line, complex mathematics models and algorithms are now being developing, checking and validating in order to select the best embryo for transfer and to help embryologist in this responsible and at the same time subjective task. In this final presentation, Dr. Diego Ezcurra will explain the latest advances in this promising area of artificial intelligence.