Recent advances in embryology

Introduction

In vitro fertilization (IVF) is a popular method for treatment of infertility owing to both male and female factors. Historically, this method was developed in order to treat infertility in women arising due to blocked fallopian tubes that impeded the fertilization of the ovum by the sperm. However, over the years this technique has become very efficient in treating infertility owing to other causes as well, including unexplained infertility.¹   However, despite phenomenal improvements in IVF methods over the past 3-4 decades, the efficiency of IVF procedures, as defined by live birth rate, is still only about 30-40%. Other problems associated with this technique include need for repeated cycles before pregnancy can be achieved, which increases emotional and financial costs, as well as constitutes additional health risks for women.²

The efficiency of IVF can be increased by transferring multiple embryos in a single cycle, but this may give rise to other problems such as multiple-pregnancies and, as a result, serious health complications for mothers and babies. There is, therefore, a strong need for transfer of a single embryo, and so the development of reliable methods of selecting embryos is the need of the hour.²

Advances in embryo selection methods

Over the past three decades embryo selection methods have evolved from the more traditional to the most technologically advanced. Some of the more recent embryo selection methods include: evaluation of pronuclear morphology, preimplantation genetic diagnosis and preimplantation genetic screening. These focus on the nuclear component of the embryo, looking for potential chromosomal abnormalities. However, despite the availability of these modern options visual assessment of embryo morphology, the most traditional method of embryo selection continues to retain its popularity.²

Visual assessment of embryo morphology by light microscopy

The assessment of embryo morphology at different developmental stages by light microscopy is the most traditional and popular method of embryo selection. Several parameters, assessed at different developmental stages, provide valuable information about the quality of embryos. Embryos can be graded according to the morphology of their pronuclei on day 1 after fertilization, the number and shape of blastomeres and degree of fragmentation on day 2 or 3, or by the morphology of the blastocyst at day 5 or 6 (Figure 1). Depending on the procedure, embryos are evaluated at one or several developmental stages.²

While this method has its advantages (low cost and ease of implementation in a clinical environment), its main disadvantage includes observer variability and lack of accuracy. The visual grading of embryos is subjective and requires significant expertise. Besides, even when such expertise is available, the technique is not always accurate: low-graded embryos often prove to have high developmental potential and can develop to term. In order to overcome these limitations, alternative method of embryo evaluation have been developed that provide more detailed information about the developmental status of the embryo and, most importantly, are quantitative and objective.²

Preimplantation genetic diagnosis and screening

Detection of inherited genetic disorders in the embryo prior to implantation is important for the success of the IVF technique. This can be done using polymerase chain reaction (PCR)-based techniques to diagnose specific genetic mutations or fluorescence in situ hybridization (FISH) to diagnose chromosomal abnormalities or to sex the embryos for patients carrying X-chromosome-linked diseases. Preimplantation genetic diagnosis can be applied to embryos at different stages: zygotes (polar body biopsy), cleavage stage embryos (blastomere biopsy) or blastocysts (trophectoderm biopsy).²

An alternative approach known as preimplantation genetic screening is used to improve IVF outcomes in mothers of advanced age, those presenting with repeated miscarriages or implantation failure, or in cases of severe male factor infertility. Current approaches in preimplantation genetic screening utilize technologies based on comparative genomic hybridization (CGH) or single nucleotide polymorphism (SNP) arrays. Both of these approaches allow the whole embryonic genome to be analyzed and are used at different embryonic stages.²

Analysis of embryo metabolism

Recent scientific evidence suggests the possibility of an association between embryo metabolism and viability. Analysis of embryo metabolism, either by the analysis of single metabolites or by a broader approach (metabolomic or protein secrotome profiling), can therefore be helpful while selecting embryos for transfer. Oxygen consumption and amino-acid turnover have proved to be reliable indicators of an embryo’s viability. Metabolomic or protein secrotome profiling is a recent approach which provides a complete picture of an embryo’s metabolism and gene expression patterns. However, although these techniques have potential value for the IVF clinics, for the moment they remain difficult to implement in a clinical environment. This is because metabolomic or secreted protein analyses involve spectroscopic/spectrometric and chromatographic techniques, which currently require expensive equipment and highly skilled staff.²

Dynamic assessment of embryonic development by time-lapse imaging

Recent advances in non-invasive time-lapse imaging techniques now allow us to follow the dynamics of embryo divisions and other fertilization-triggered events and correlate them with the developmental potential of the embryos. In the last two years there have been two studies employing this technique in a very different way. A team at Stanford University has shown that the timing and synchrony of the first two embryonic divisions are predictive of developmental potential of human embryos (Figure 2). The authors reported that embryos with a very long first cytokinesis, with a prolonged or an atypically short interval between first and second division or with highly asynchronous divisions of two-cell blastomeres, fail to reach the blastocyst stage. The findings of this study are in concordance with previous observations, in which timely pronuclear formation and subsequent first cleavage correlated with higher quality of human embryos.

The method described by Wong et al. involves timelapse imaging of embryos every 5 min for several hours (from 1- to 4-cell stage). In comparison, the method established by Ajduk et al. requires a much shorter period of recording (2.5 hours for mouse embryos) but with images taken every 10 seconds.

Ajduk A, Zernicka-Goetz M. F1000 Biology Reports 2012, 4:11

Another team at University of Cambridge working collaboratively with teams at Oxford and Cardiff Universities developed a completely different approach. Their work showed that fertilization of eggs triggers abrupt, repetitive cytoplasmic movements that correlated with Ca²⁺ oscillations (also triggered by sperm) and depended on the functionality of the cytoskeleton. Moreover, the cytoplasmic flows were predictive of the developmental potential of the embryos. Embryos that showed very frequent increases in cytoplasmic movements (indicating very frequent Ca²⁺ oscillations) and low cytoplasmic speeds in the intervening periods (reflecting low quality of the actin cytoskeleton) were three-fold less successful in developing to term than embryos displaying average values of these parameters. Both methods have yet to be tested in a clinical environment and subjected to randomized controlled trials to demonstrate improved live birth outcomes.²

Advances in embryo transfer

Embryo transfer (ET) is the crucial last step in IVF. This procedure involves delicate placement of the embryo(s) at the proper location near the middle of the endometrial cavity with minimal trauma and manipulation. The ultimate goal of a successful ET is to deliver the embryos atraumatically to the uterine fundus in a location where implantation is maximized.¹

The transfer of embryos can be accomplished in several different fashions including transfallopian (ZIFT), transmyometrial and transcervical ways. The transcervical approach is preferred route for the majority of ETs.¹   Traditionally ET was performed blindly. The internal cervical os was palpated, the catheter was advanced till the fundus was felt and then withdrawn for 1 or 2 cm and the embryo(s) were positioned. Alternatively the clinician advanced the inner catheter after feeling the resistance of the internal os by 3-4 cm based on previous measurement of the uterine cavity. However, tactile assessment of ET catheter placement is unreliable, especially in inexperienced hands, and unavoidable sub-endometrial transfers and accidental tubal transfers frequently occur. Hence, there is an urgent need for additional means to ensure the proper deposition of the transferred embryos.³

Ultrasound-guided ET

Ultrasound-guided ET is a simple modification of the clinical touch method of ET. This modification uses a two-dimensional (2D) transabdominal ultrasound to visualize the anatomy of the pelvic structures. A 3D ultrasound provides better results. While the blind ET procedure depends very much on the experience of the performer, thereby giving inconsistent results in different hands, ultrasound-guided ET allows for standardization of the transfer technique.3

Scientific evidence shows that patients undergoing ET under ultrasound-guidance have a significantly higher likelihood of achieving a live birth, ongoing pregnancy and clinical pregnancy than patients undergoing the standard ‘clinical touch’ ET. Ultrasound-guided ET allows for embryo(s) to be placed lower in the uterine cavity (rather than at the fundus) that may improve pregnancy rates. Coroleu et al. found that embryos should be positioned 15-20 mm from the fundus endometrial surface rather than performing high fundal placement in order to improve implantation rates. Pop et al. found that for every additional mm embryos are deposited away from the fundus, the odds of clinical pregnancy increased by 11%.³

Mock or “dummy” ET

Researchers have demonstrated that even light stimulation of the fundus during ET can produce fundocervical waves that may displace the embryo towards the cervix. The use of soft-tipped catheters, ultrasound, or fixed distance transfers minimize inadvertent stimulation of the fundus, and have been linked to improved pregnancy rates with IVF-ET.⁴

It is now a common practice to perform a mock or “dummy” ET before the start of an IVF cycle. A mock ET ensures that entry into the uterine cavity is possible and enables the measurement of the uterocervical angle and depth of the endometrial cavity. Thereby difficulties, which may lead to a traumatic ET or even failure of ET, can be anticipated and avoided.⁴   Mock ET improves pregnancy rate (45%) and implantation rate (20.6%) per embryo.⁵   However, a mock transfer remote from the actual ET is done under different circumstances and may not be reflective of actual conditions encountered on the day of ET. Performing a mock ET immediately before the actual ET could overcome this problem.⁴

Embryo afterloading

In the afterload technique the “dummy” ET was performed immediately before the actual ET; however, additional trauma by the passage of two separate catheters was avoided by placement of an empty catheter at, or just past, the internal cervical os. The inner sheath was withdrawn, and a second inner sheath containing the embryos was passed. This gave the provider the benefit of an immediate mock transfer while minimizing manipulation of embryos and possibly reducing trauma to the endometrium thus improving pregnancy rates.⁴

A retrospective analysis of 127 ETs done during a 1-year period showed that the implantation rate per embryo was higher, although not significantly, in the afterload group (Figure 3), while the clinical pregnancy rate in the afterload group was significantly higher than in the direct transfer group (P=.06) (Figure 3).

Conclusion

Over the past three decades embryo selection methods have evolved from the more traditional to the most technologically advanced. Some of the more recent embryo selection methods include: evaluation of pronuclear morphology, preimplantation genetic diagnosis and preimplantation genetic screening. These focus on the nuclear component of the embryo, looking for potential chromosomal abnormalities. Others (e.g. analysis of metabolites, secreted proteins or cytoplasmic flows) concentrate on the cytoplasmic component, analysing the quality of embryo metabolism, the cytoskeleton or Ca²⁺ homeostasis. We believe that the ultimate selection method should combine these approaches. Recent publications suggest that a shorter period between in vitro fertilization and ET may be beneficial, as prolonged in vitro culture of embryos alters their epigenetic modifications and gene expression.

The technique of ET can have a great impact on the outcome of IVF treatment. Transcervical ET is a blind procedure and its success largely depends on the experience of the clinician. Ultrasound-guided ET improves outcome. However, the outcome of the IVF cycle depends on several other factors as well including ease of ET, blood on the transfer catheter, type of catheter used and whether or not inadvertent stimulation of the uterine fundus has occurred during ET. Many IVF programmes therefore perform a ‘mock’ ET prior to the treatment cycle to determine the most suitable catheter and technique for transfer. However, a mock transfer remote from the actual ET is done under different circumstances and may not be reflective of actual conditions encountered on the day of ET. Performing a mock ET immediately before the actual ET by the “afterloading” technique could circumvent this problem. Embryo afterloading is a refinement of standard ET and may improve clinical pregnancy rates.

References

1. Wu B. Advances in Embryo Transfer. Available at : http://cdn.intechopen.com/ pdfs-wm/32051.pdf. Accessed on 10/12/2015.
2. Ajduk A, Zernicka-Goetz M. Advances in embryo selection methods. F1000 Biology Reports 2012, 4:11.
3. Yehia M. ET: does ultrasound guidance make a difference? Middle East Fertility Society Journal 2006;11(3):173-82.
4. Neithardt AB, Segars JH, Hennessy S, James AN, McKeeby JL. Embryo afterloading: a refinement in embryo transfer technique that may increase clinical pregnancy. Fertil Steril. 2005 Mar;83(3):710-4.
5. Sharif K, Afnan M, Lenton W. Mock embryo transfer with a full bladder immediately before the real transfer for in-vitro fertilization treatment: the Birmingham experience of 113 cases. Hum Reprod. 1995 Jul;10(7):1715-8.