Physiology and culture of the human blastocyst

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Abstract

The human embryo undergoes many changes in physiology during the first 4 days of life as it develops and differentiates from a fertilized oocyte to the blastocyst stage. Concomitantly, the embryo is exposed to gradients of nutrients within the female reproductive tract and exhibits changes in its own nutrient requirements and utilization. Determining the nature of such nutrient gradients in the female tract and the changing requirements of the embryo has facilitated the formulation of stage-specific culture media designed to support embryo development throughout the preimplantation period. Resultant implantation rates attained with the culture and transfer of human blastocysts are higher than those associated with the transfer of cleavage stage embryos to the uterus. Such increases in implantation rates have facilitated the establishment of high pregnancy rates while reducing the number of embryos transferred. With the introduction of new scoring systems for the blastocyst and the non-invasive assessment of metabolic activity of individual embryos, it should be possible to move to single blastocyst transfer for the majority of patients.

Introduction

The mammalian preimplantation embryo represents an intriguing challenge to the biologist due to its changing physiology as it develops and differentiates. Specifically, the fertilized oocyte behaves metabolically in many ways like a quiescent tissue, while just a few days later the blastocyst is more akin to an invasive tumor prior to implantation (Gardner, 1998a). These changes in physiology facilitate the successful development of the embryo as it proceeds along the female reproductive tract and prepares to implant in the uterine endometrium. Taking into account such dynamics in physiology has lead to the development of sequential culture systems capable of supporting the development of viable human blastocysts.

Section snippets

Human embryo physiology

In the human, fertilization occurs in the ampullary-isthmic region of the fallopian tube and the subsequent embryo is then transported along the tube into the uterus with the help of the tubal epithelial secretions, cilia, and peristaltic contractions of the tubular musculature. The first 3–4 days of embryonic development from syngamy to the morula stage occurs in the fallopian tube (Croxatto et al., 1972). For the majority of this time the cells of the embryo (blastomeres) remain unattached

Nutrient requirements and energy metabolism

Concomitant with changes in physiology, the mammalian embryo undergoes changes in nutrient requirements and utilization during the preimplantation period. The human embryo, like that of several other mammalian species, has an initial preference for pyruvate over glucose as a nutrient (Hardy et al., 1989a). However as development proceeds the human embryo exhibits an increasing capacity to utilize glucose and by compaction glucose is the main carbohydrate utilized. The reason for the change in

Nutrients available to the embryo

Within the female reproductive tract, the human embryo is exposed to a gradient of carbohydrates (Table 2). Amino acids such as glycine and taurine present at relatively high levels in oviduct fluid are those found to aid cleavage stage embryo development. Therefore, the nutrients available to the embryo as it develops and passes along the female reproductive tract appear to mirror the stage-specific requirements of the embryo.

Culture systems for the human embryo

The growing data on the composition of human female reproductive tract fluids and from embryo physiology studies has led to the proposition that extended embryo culture should take place in more than a single medium formulation (Gardner and Leese, 1990). To this end, sequential media have been developed, tested extensively on animal models and subsequently used clinically (Gardner and Lane, 1997, Gardner and Lane, 1998, Menezo et al., 1998).

Clinical use of sequential media

Use of sequential culture systems was initially offered to patients with either a good response to gonadotropins (Gardner et al., 1998a, Gardner et al., 1998b) or with >4 eight-cell embryos on day 3 (Milki et al., 1999). This approach for good prognosis patients led to a significant increase in implantation rates and facilitated the establishment of high pregnancy rates (70%) with a concomitant reduction in the number of embryos transferred. Therefore, the use of sequential culture media led a

What is the rate-limiting factor at implantation; the embryo or the endometrium?

A number of conceptual models have been developed to examine the variation in outcomes following embryo transfer. Gardner et al. (2000c) used a statistical model to determine the contribution of embryo quality and endometrial receptivity in 266 IVF patients (mean age of 34) and 122 oocyte donor recipients (mean age of 41) having blastocysts transferred. The analysis revealed that blastocyst competence was similar in both groups of patients. However, uterine receptivity was improved in oocyte

Blastocyst transfer: a panacea for all ills?

It would be unwise to suggest that blastocyst culture and transfer represents a panacea for all clinics and all patients as, although many clinics have experienced increased success with extended culture, others have reported no benefit (Coskun et al., 2000). Before extended culture should be considered, all aspects of clinical and laboratory procedures need to be optimized. Should problems exist either in patient stimulation protocols or within the laboratory, extended culture may only

Conclusions

The successful implementation of blastocyst culture and transfer is facilitated by the optimization of both clinical and laboratory procedures that are readily monitored in either good prognosis patients or oocyte donors. In such patient groups, blastocyst rates of 50% can be readily attained with subsequent implantation rates of at least 40%. Should such rates not be forthcoming, then one must analyze stimulation and luteal support regimens as well as examining laboratory protocols and quality

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      Embryos were cultured at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. Blastocysts were scored according to the grading system of Gardner and Schoolcraft (Gardner et al., 2002), and were considered eligible for cryopreservation on day 5 or day 6 if they qualified as full (B3) or expanded (B4–5) blastocysts with a type A–C inner cell mass, type A–C trophectoderm, or both. Blastocysts with a type ‘C’ inner cell mass and a type ‘C’ trophectoderm were not cryopreserved regardless of their degree of expansion.

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