Human Reproduction E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 663

Veröffentlichungsjahr: 2016

Beschreibung

In vitro fertilization (IVF) and other assisted reproductive technologies (ART) have become a significant part of human reproduction, with already one in 50 children worldwide being born through ART and the demand steadily increasing. To accommodate the various kinds of infertility problems, new methods have been developed to increase IVF and ART success rates and it has also become possible to treat sperm, eggs, and embryos in culture to improve reproductive success, to increase the health state of an embryo, and to prevent disease in the developing child. Human Reproduction: Updates and New Horizons focuses on recent developments and new approaches to study egg and sperm cells and embryo development, as well as addressing the increasing demand for IVF and ART to overcome infertility problems of various kinds that are encountered by an increasing number of couples worldwide. The book includes 10 chapters written by experts in their specific fields to provide information on sperm selection techniques and their relevance to ART; In vitro maturation of human oocytes: current practices and future promises; Molecular biology of endometriosis; Novel immunological aspects for the treatment of age-induced ovarian and testicular infertility, other functional diseases, and early and advanced cancer immunotherapy; Mitochondrial manipulation for infertility treatment and disease prevention; Novel imaging techniques to assess gametes and preimplantation embryos; Clinical application of methods to select in vitro fertilized embryos; New horizons/developments in time-lapse morphokinetic analysis of mammalian embryos; The non-human primate model for early human development; Cytoskeletal functions, defects, and dysfunctions affecting human fertilization and embryo development. This book will appeal to a large interdisciplinary audience, including researchers from both the basic science and medical communities. It will be a valuable reference for IVF clinicians, patients and prospective patients who are considering ART procedures, embryologists, cell biologists and students in the field of reproduction.

Leseprobe

Table of Contents

Title Page

Copyright

List of Contributors

Chapter 1: Sperm Selection Techniques and their Relevance to ART

1.1 Introduction

1.2 Need of Sperm Selection in ART

1.3 Methodology of Sperm Selection

1.4 Electrophoretic Sperm Separation

1.5 Zeta Test

1.6 Microelectrophoresis Sperm Selection

1.7 Raman Spectroscopy

1.8 Hyaluronic Acid Binding Assay

1.9 Future Perspective

References

Chapter 2: In Vitro Maturation of Human Oocytes: Current Practices and Future Promises

2.1 Introduction

2.2 Clinical Indications for IVM

2.3 Ovarian Stimulation Approaches for the Retrieval of Immature Oocytes

2.4 Maternal Conditions that may Influence IVM

2.5 Follicular Origins of Immature Oocytes for IVM

2.6 Clinical Safety of IVM

2.7 Concluding Remarks towards the Optimization of IVM

References

Chapter 3: Molecular Biology of Endometriosis

3.1 Introduction

3.2 Brief Background

3.3 Genetic Basis of Endometriosis

3.4 Molecular Mechanisms of Endometriosis

3.5 Molecular Etiopathological Basis of Endometriosis: Leads in Genomics Era

3.6 Molecular Etiopathological Basis of Endometriosis: Leads in the Post-Genomics Era

3.7 Future Targets

Acknowledgments

Conflicts of Interest

References

Chapter 4: Novel Immunological Aspects for the Treatment of Age-induced Ovarian and Testicular Infertility, Other Functional Diseases, and Early and Advanced Cancer Immunotherapy

4.1 Introduction

4.2 Ovarian Infertility

4.3 Novel

In Vitro

Proposals for Ovarian Infertility Treatment

4.4 Novel

In Vivo

Proposal for Ovarian and Testicular Infertility Treatment

4.5 Systemic Treatment of Other Functional Diseases by Tissue Rejuvenation

4.6 Advantages of Local and Systemic Use of Honey Bee Propolis and Cayenne Pepper

4.7 The Promise of Pyramid Healing Systems

4.8 Raw Shiitake Causes Early Neoplasia Regression and Malignancy Recurrence Prevention

4.9 Immune Modulation for the Treatment of an Advanced Cancer

4.10 Advanced Ovarian Cancer Regression Case Report

4.11 Discussion

4.12 Conclusions

Abbreviations

Competing Interests

Author Contribution

References

Chapter 5: Mitochondrial Manipulation for Infertility Treatment and Disease Prevention

5.1 Introduction

5.2 The Roles of Mitochondria in Fertilization, Embryonic Development, and Disease

5.3 The Genetics of Mitochondria and Mitochondrial Diseases

5.4 Ooplasmic Transfer to Treat Infertility

5.5 Pronuclear Transfer to Achieve Pregnancy

5.6 Germinal Vesicle Transfer to Restore the Viability of Oocytes

5.7 Mitochondrial Diseases and Prevention of their Inheritance

5.8 Mitochondrial Replacement by Transferring Pronuclei and MII Spindle

5.9 Discussion

Acknowledgments

References

Chapter 6: Novel Imaging Techniques to Assess Gametes and Preimplantation Embryos

6.1 Introduction

6.2 Light and Impact on Mammalian Gametes and Embryos

6.3 Novel Imaging Approaches for Gametes and Embryos

6.4 Conclusion

References

Chapter 7: Clinical Application of Methods to Select In Vitro Fertilized Embryos

7.1 Introduction

7.2 Morphological Assessment

7.3 Genomic and Transcriptomic Analysis

7.4 Analysis of Conditioned Culture Medium

7.5 Summary

References

Chapter 8: New Horizons/Developments in Time-Lapse Morphokinetic Analysis of Mammalian Embryos

8.1 Introduction

8.2 Utilization of Time-Lapse Morphokinetics in Mammalian Embryos: A Historical Perspective

8.3 What is TLM?

8.4 What are the Benefits of TLM?

8.5 Application of TLM in Human ART Practice

8.6 The Possible Utilization of TLM Analysis in Aneuploidy Detection

8.7 Expected Contributions of TLM Technology in the Future of Mammalian Embryology

References

Chapter 9: The Non-Human Primate Model for Early Human Development

9.1 Introduction

9.2 Why Primate Models Are Critical to Understanding Human Development and Subfertility

9.3 NHP Model of Assisted Reproductive Technology (ART)

9.4 NHP Model of Early Embryo Development

9.5 Research Perspective on NHP Embryo Development

9.6 Summary

References

Chapter 10: Cytoskeletal Functions, Defects, and Dysfunctions Affecting Human Fertilization and Embryo Development

10.1 Introduction

10.2 Components of the Cytoskeleton and their Important Functions in Reproductive Biology

10.3 The Role of the Cytoskeleton in Oocyte Maturation

10.4 Maturation Failures and Oocyte Aging

10.5 Fertilization and First Mitosis/Cell Division

10.6 Cellular Differentiation/Polarization During Pre-Implantation Embryo Development/Compaction Stage

10.7 Perspectives and Future Directions

References

Index

End User License Agreement

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Guide

Table of Contents

Begin Reading

List of Illustrations

Chapter 1: Sperm Selection Techniques and their Relevance to ART

Figure 1.1 Apoptotic sperm are labeled by annexin V magnetic beads. A magnetic field separates the apoptotic sperm.

Figure 1.2 Separation of motile sperm using microfluidics.

Figure 1.3 Schematic diagram showing the apparatus for the electrophoretic sperm separation.

Figure 1.4 Diagram of sperm selection using the Zeta test. Negatively charged mature sperm is adhered to the positively charged tube surface, while immature sperm remain suspended in the media.

Figure 1.5 Schematic representation of microelectrophoresis unit.

Figure 1.6 A typical micro-Raman spectroscopy setup as utilized in the analysis of sperm.

Chapter 3: Molecular Biology of Endometriosis

Figure 3.1 Knowledge-based construction of the pathways-network of transcription factors putatively associated with the pathogenesis of endometriosis, obtained from functional analysis of co-expressed genes. CLOCK, ESR1, and MYC genes (shown inside

blue dotted rectangle

) appear to be involved in pathogenesis of endometriosis. Adapted from Ref. 154.

Figure 3.2 Pathways map derived from the enrichment analysis of differentially regulated genes between eutopic endometrium of subfertile patients with ovarian endometriosis in both proliferative and secretory phase of menstrual cycle highlighting important cellular processes getting involved in endometriosis. This Figure illustrates that molecular processes like angiogenesis (STAT signaling being the major mover), cell proliferation and apoptosis (MAPK signaling being the major mover) are affected in the eutopic endometrium of patients having ovarian endometriosis. Three major regulators – HIF1A, EGR1 and cFos (shown as

asterisks

) – are reportedly affected in endometroisis (142, 154, 160). Tables 3.3–3.5 highlight many of these genes in the process of development of endometriosis.

Figure 3.3 Cellular factors known to be associated with the pathways to neoplasm and tumorigenicity, some of which are reportedly dysregulated in eutopic and ectopic tissues in endometriosis as shown in

red

bullets. Interestingly, several studies on genomics expression, epigenetic changes and proteomics profile in endometriosis converge on the proposed involvement of these factors. For details, see Tables 3.3–3.7 and the text.

Figure 3.4 Immunopositivity of annexin A2 (a and b), HSP90 (c and d), PDGFRa (e and f), and Tubulin A (g and h) in eutopic endometria of subjects with diagnosed stage IV ovarian endometriosis during secretory phase of menstrual cycle showing their higher expression as compared to normal endometrium. Bars: 50 µm (c and d), 60 µm (h), and 75 µm (a, b, and e–g). Adapted from Ref. 196.

Figure 3.5 Venn diagram showing overlap of differentially expressed miRNAs in four different studies: Study A (244), Study B (278), Study C (279), and Study D (246). The four common miRNAs in these studies were miR-29c, miR-100, miR-200a, miR-200b. Some functional details of these miRNAs are given in Table 3.7. Adapted from Ref. 246.

Chapter 4: Novel Immunological Aspects for the Treatment of Age-induced Ovarian and Testicular Infertility, Other Functional Diseases, and Early and Advanced Cancer Immunotherapy

Figure 4.1

Immune type cells influence commitment of OSCs in adult human ovary (age 32 years, midfollicular phase)

. (a) Primitive CD14 MDC (green asterisk) associates with a small OSC (yellow asterisk and dotted circle) accompanying origination (green arrowhead) of a larger germ cell (red asterisk and dashed line) by asymmetric division of OSC (red arrowhead). (b) A serial section shows that asymmetric division is also accompanied by CD8 T cell (white asterisk) entering germ cell and exhibiting extensions (white arrowhead). (c) Divided primitive CD14 MDCs (green asterisks) accompany (green arrowheads) symmetric division (meiosis I cytokinesis) of germ cells (red asterisks) in the TA (ta) and germ cells moving (arrow) into the adjacent upper ovarian cortex (uoc). Inset shows a blood venule in the upper ovarian cortex with Thy-1 differentiation protein expression by vascular pericytes (arrow) and venule lumen (vl) containing a germ cell (red asterisk). (d) Germ cell transport in the upper ovarian cortex is associated with an attached activated (DR+) MDC (green asterisk and dotted lines) releasing DR+ cytoplasmic particles (green arrows) that accumulate at the surface of the germ cell nucleus (arrowhead). (e) Endothelial cells (en and open arrows) of a venule in the upper ovarian cortex exhibit MHC-I expression, which is not expressed by associated migrating (red arrow) germ cell (asterisk). See text for additional details. Adapted from Bukovsky et al. (1995b), with permission: © Blackwell Publishing, Oxford, UK.

Figure 4.2

Formation of fetal germ cells, granulosa cells, and follicular development in midpregnancy human fetal ovary.

(a) CD14 MDC interacts (arrowheads) with an OSC (yellow asterisk and dotted circle) prior to the asymmetric division. (b) Numerous MHC-I depleted fetal germ cells (blue asterisks and dashed circles) originating by asymmetric divisions (arrowheads) from MHC-I+ OSCs (yellow asterisks and dotted circles). (c) Asymmetric division (blue arrowhead) is followed by a symmetric division of MHC I depleted germ cells (red asterisks and arrowhead). This is followed by the development of an ameboid shape moving germ cell (mgc – dashed line, no hematoxylin counterstain) entering adjacent ovarian cortex. Asymmetric division is accompanied by CD8 (d) and DR+ (e) T cell. (f) CD14 MDC (arrowhead) accompanies symmetric division of germ cell during meiosis I telophase. (g) Development of primitive granulosa cells (pgrc, note lower CK expression) from ovarian stem cells between mesenchymal cell cords (mcc). (h) DR+ MDC accompany (arrows) fetal growing follicle (gf) but not the resting follicle (rf). Inset shows association of Thy-1+ pericytes (arrowhead) with a growing but not resting follicles. Bar in a for a–f. Adapted from Bukovsky et al. (2005a) with permission: © Springer US.

Figure 4.3

Origin, meiosis I, and migration of human adult germ cells, and follicular renewal in a 28-year-old women (F28).

(a) Dual color IHC of asymmetrically dividing OSC with CK+ (blue dashed line) OSC daughter and PS1+ (red dashed line) germ cell daughter. CK+ OSC daughter chromosomes (white arrowhead) move to the OSC end during mitotic OSC anaphase. Germ cell chromosomes (white arrows) duplicate by DNA replication (red and blue arrows) and exhibit sister chromatid crossover (orange arrows) during meiosis I prophase. White asterisk and dotted circle indicate PS1+ putative CD8/DR+ suicidal T cell within the germ cell (see Figure 4.1b, 4.2d, 4.2e and Bukovsky et al., 2001a). (b) In the TA (ta) the symmetrically dividing germ cell exhibits strong nuclear (asterisks) PS1 expression, which accompanies the meiosis I telophase. Arrow indicates a germ cell moving from the TA to the upper ovarian cortex (uoc). Inset shows a detail of chromosomal crossover (orange arrows) from panel A. Red and blue arrows indicate interacting sister chromatids. (c) Germ cell with a diminution of nuclear and increase of cytoplasmic PS1 staining. It begins to enter (arrowheads) the vein in the upper ovarian cortex. (d) Early stage of new primary follicle formation with ZP (blue color) expression of a small oocyte captured by the CK+ (brown color) granulosa cell nest. See details in the text. Adapted from Bukovsky et al. (2004): © Antonin Bukovsky.

Figure 4.4

Follicular renewal in adult human ovary and intravascular degeneration of germ cells unattended with granulosa cells.

(a) Ovarian vein in the lower ovarian cortex lined by endothelial cells (en) and CK+ granulosa nest wall (gnw). In the vein lumen (vl) the granulosa nest wall extends a granulosa nest arm (gna) capturing the circulating oocyte (co). (b) Granulosa nest (gn) during formation of the new primary follicle with captured oocyte. Granulosa cells penetrate the ooplasm (red arrowheads) during the primary Balbiani body (asterisk) formation adjacent to the oocyte nucleus (on). CK+ granulosa nest particles (yellow arrowheads) are already dispersed within the oocyte, which still exhibits oocyte tail (ot) outside of the new primary follicle. (c) Growing preantral follicle (dashed line circle), with granulosa cells (grc) and oocyte (o) with ZP expression at the oocyte surface (arrow). (d) Degenerating oocyte in a medullary vein from the same ovary as in panel C exhibits a strong cytoplasmic ZP expression. (e) Heavily ZP+ degenerating oocyte from 28-year-old woman found in the extra ovarian (uterine ectocervix) vein of a patient with follicular renewal shown in panels a and b. Panels A and B adapted from Bukovsky et al. (2004): © Antonin Bukovsky; panels C–E adapted from Bukovsky et al. (2008b), with permission: © Elsevier/North-Holland Biomedical Press.

Figure 4.5

Cyclic formation of OSC, granulosa cells nests, and presence of new and resting primary follicles during midfollicular phase.

(a) Tunica albuginea (ta) fibroblasts (fb) type OSC precursors with CK immunoexpression (brown). Two cells in mesenchymal-OSCs epithelial transition (fb/osc) are apparent. (b) Appearance of OSCs (osc) is associated with CK depletion (-fb). (c) Formation of CK+ granulosa cell nests is initiated by a layer of OSC (white arrows) above upper ovarian cortex (uoc). This is overgrown by a developing flap of TA (ta flap or taf in insert) resulting in a bi-layered osc cord (black arrow). Inset shows two layers of the OSC channel. (d) Detail of OSC flap with CK+ fibroblast type OSC precursors (fb/osc), and OSC development above the upper ovarian cortex (arched arrow). Arrowhead indicates the flap content of OSCs. (e) A parallel section to (D) showing numerous DR+ MDC (asterisks) in the TA flap. Note DR expression in early OSC (arrow). (f) Detail of OSC-cord from panel C shows CK+ epithelial cord. (g) OSC flap (red arrowhead) over a segment of TA (dashed line) covered by OSC layer (red arrow). The OSC cord-derived granulosa cell clusters (black arrows) fragment into granulosa cell nests (black arrowheads). Dashed line indicates a segment of TA covered by OSC epithelium. (h) Granulosa cell nests (black arrowheads) move by stromal rearrangements (arched arrow) to the lower ovarian cortex (loc) and form new primary follicles (white arrowhead) containing ZP+ oocytes. (i) Lower ovarian cortex (loc) with new primary (right panel segment) and resting primary follicles (left). Right inset shows the presence of primary Balbiani bodies. Left inset shows lack of Balbiani bodies. Bar in (a), for (a and b), bar in (f) for (d–f). Adapted from Bukovsky et al. (2004): © Antonin Bukovsky.

Figure 4.6

in vitro

developing oocytes are supplied with meiotically nonfunctional organelles from fibroblasts or satellite cells.

Time lapse photography shows that early developing oocytes (o, panel (a) are low in optically dense cytoplasmic organelles (white open arrow). They can be joined (arrowhead) by fibroblast-like cells (fb), providing additional organelles. Such fibroblast-type cells initially show optically dense organelles close to the nucleus (black solid arrow), but not in the arm extended toward the oocyte (white solid arrow). Within 10 min (panel (b), however, the optically dense organelles are apparent in the extended arm (solid black arrow) and within adjacent oocyte cytoplasm (black arrowhead) and distant oocyte regions (black open arrow). At 4h 25 min (panel c), however, the fibro-oocyte (fbo) hybrid is formed and regressing oocyte (ro) exhibits depletion of organelles (arrow) accumulated by the fibroblast (arrowhead). Alternatively, the developing oocytes (o, panel d) deficient in cytoplasmic organelles (white arrow) exploit the satellite cells (s), which are produced by the oocytes themselves. The oocyte is supplied by suicidal satellite cell by a tube like ring canal (black arrowhead; see inset). In panel e the oocyte exhibits enhanced content of the optically dense organelles (black arrow) and the ring canal draining the satellite disappears (white arrowhead – see inset). The satellite cell size is reduced (dashed line) and the perinuclear space is altered (compare with panel d). Oocytes enriched by satellites' organelles (panel f) exhibit good morphology [200 µm size, germinal vesicle (gv), and thick zona pellucida (zp)], but are unable to resume meiosis II due to the lack of meiotically functional organelles provided by secondary Balbiani body derived from granulosa cells

in vivo

. Bar in a for a–e. Panel c reprinted from Bukovsky and Caudle (2012): © Antonin Bukovsky. Other panels adapted from Bukovsky (2011b), with permission: © Wiley-Liss, Inc.

Figure 4.7

Time lapse video of oocyte reconstruction in secondary OSC culture

(a) Early developing cell with a cytoplasmic tail (arrowhead). (b) Multiple cytoplasmic eruptions (arrowheads). (c) Development of the 50 µm oocyte-like cell (yellow arrowheads indicate cell surface, red arrowhead a polar body). Time in min':sec''. Reprinted from Bukovsky and Caudle (2012): © Antonin Bukovsky.

Figure 4.8

ZP and CK expression in ovarian follicles

. (a) Oocytes in resting primary follicles lack ZP3 expression, but express ZP1, ZP2, and ZP4 (Bukovsky et al., 2008b). (b) ZP3 is expressed in the oocyte of a growing preantral follicle. (c) Double color IHC for CK (brown) and ZP (blue) expression in a growing preantral follicle. CK is expressed in granulosa cells (GrC) but no secondary Balbiani body is present in the oocyte. Oocyte surface expresses ZP (blue arrowhead). (d) Double color IHC for CK (blue) and ZP (brown) expression in a small antral follicle. CK is expressed in granulosa cells (GrC) and in the oocyte secondary Balbiani body (blue arrowhead). Oocyte surface expresses ZP (orange arrowhead).

Figure 4.9

ZP3 expression by OLCs in IVM treated OSC cultures is stolen by fibroblasts

. (a) OLCs in untreated OSC cultures show week nuclear ZP3 expression, which is absent in accompanying satellite cells (SC) and fibroblasts (FB). Arrowheads indicate tube like ring canals between OLC and SC; arrows indicate bindings of FBs to the OLC. (b) After hCG treatment the OLC exhibits strong nuclear and surface (black arrowhead) ZP3 expression, which is also present in accompanying SC (white arrowhead). The ZP3 expression is stolen by FBs (red arrowheads), leaving the surface of OLC ZP3 depleted (open arrowhead). (c) The OSC culture from a 30-year-old POF female was IVM (FSH+hCG) pretreated and fertilized with the husband's sperm. The phase contrast (PhC) image from a live culture shows that the sperm are associated with fibroblasts instead with OLCs.

Figure 4.10

Propolis tincture preparation for the mouth and systemic use, and local effects for the hair, varicose veins, and teeth.

(a) Measurement of 30 ml 40% alcoholic distillate. (b) Over layered propolis tincture for the local teeth treatment and dilution of the poured back propolis tincture for the systemic propolis treatment (c). (d) Developing frontal alopecia (arrow) before the local propolis treatment (back to front hair orientation) - note association of hair color depletion (dashed line circle) with developing alopecia. (e) Persisting unchanged frontal alopecia (arrow) (back to front hair orientation) – note restoration of the original hair color (compare with panel d) but persistence of color depletion in untreated coat sides (yellow arrowhead). (f) Hair condition in normal (side to side) hair orientation. (g) Recent hair appearance showing further improvement of hair condition, including color regeneration in propolis treated sides (red arrowhead). (h) Shrinking (blue arrows) and regressed (yellow arrows) varicose veins on the legs after the local propolis treatment. (i) Upper teeth row after professional dental cleaning and (j) heavy propolis deposits (arrows) thereafter on the densely brushed teeth. (k) Residual propolis attachments four days later. (l) Teeth status after 5 months of propolis treatment showing no propolis binding without any dental brushing, which indicates well-regenerated dental enamel. Arrowheads indicate a diminution of dental fissure (compare panel i). Numbers in blue rectangles indicate the image collection dates.

Figure 4.11

A Pyramid above the bed.

Self-constructed pyramid consisting of 6.4 mm thick copper round, with a 53 cm long base and 49 cm long side cooper rounds. Reprinted from Bukovsky, 2016.

Chapter 5: Mitochondrial Manipulation for Infertility Treatment and Disease Prevention

Figure 5.1 The mitochondrial structure and functions. ATP is mainly produced through the oxidative phosphorylation (OXPHOS) pathway in mitochondria, where respiratory chain complexes I-IV act in coordination to create a proton gradient that drives ATP production by the complex V. The subunits of the respiratory chain complexes are encoded by nuclear and/or mitochondrial genes. Electrons flow from complex I and II to complex III through coenzyme Q (CoQ), from complex III to cytochrome c (Cyt. c), and from cytochrome c to complex IV, where four electrons and protons are used to reduce O

2

to H

2

O. Although OXPHOS can leak electrons, which may generate free radicals, such as reactive oxygen species (ROS; O

·

2

−

), the free radical production is largely modulated by the rate of the electron flow.

Figure 5.2 The protocol for ooplasmic transfer.

Figure 5.3 Comparison of the three nuclear transfer protocols: germinal vesicle transfer, metaphase II spindle transfer and pronuclear transfer.

Chapter 6: Novel Imaging Techniques to Assess Gametes and Preimplantation Embryos

Figure 6.1 Representative images an (a) MII mouse oocyte with visualization of the meiotic spindle, (b) human sperm with birefringence and a vacuole, and (c) human oocyte zona pellucida showing three layers. (b) Adapted from Gianaroli et al. 2010, (c) adapted from Pelletier et al. 2004).

Figure 6.2 Representative images of rhesus oocytes with varying distribution of mitochondria (a, a', a') and (b). Rhesus 2PN zygote, (c) 1-cell, and (d) 2-cell embryo and mitochondrial distribution using Multiphoton image. Imaging required use of a fluoroprobe, but was compatible with continued embryo development (images adapted from Squirrell et al. 2003).

Figure 6.3 Representative images of a cleavage stage mouse embryo imaged with third (THG) harmonic generation (a). Image a' is the same embryo stained for lipids, which co-localized with the THG signal (a') (images adapted from Watanabe et al. 2010). Second harmonic generation imaging (SHG) can display structures such as the meiotic spindle (arrows; b, b', b') (images adapted from Thayil et al. 2011).

Figure 6.4 Representative images of a mouse GV-intact and MII oocyte imaged using Fourier transformed infrared. (a) GV intact oocyte and (a') MII oocyte; (b) chemical map of the integrated area under the amide bond of a GV oocyte and (B') MII oocyte; (c) CH

2

and CH

3

stretching region of a GV and (c') MII oocyte; (d) ester carbonyl band of a GV and (d') MII oocyte. IR imaging has been used to compare compositional differences, such as lipid distribution, between mouse oocytes of varying maturational status (images adapted from Wood et al. 2008).

Figure 6.5 Representative Raman microspectroscopy images of human sperm and a mouse oocyte. (a') brightfield image of human sperm and (a') chemical map constructed from Raman imaging of the same sperm using a cluster analysis from 400–3400 cm

−1

, showing nucleus (green), neck (red) and midpiece (yellow). (b) Raman imaging of a mouse oocyte and chemical map constructed from cluster analysis from 500–1800 cm

−1

showing zona pellucida (green), subcortical (red) and central (pink) zone of cytoplasm. Raman imaging has been used to examine compositional content and changes in gamete DNA integrity and oxidative damage (images adapted from Bogliolo et al. 2013, Meister et al. 2010).

Figure 6.6 Representative CARS images of mouse GV-intact oocytes demonstrating lipid droplet distribution (images courtesy of J. Jasensky).

Figure 6.7 Representative images optical quadrature microscopy (OQM) of mouse embryos. OQM has been used to reliably count blastomeres compared to traditional staining/counting approaches (images adapted from Warger et al. 2007, Newmark et al. 2007).

Figure 6.8 Representative images of Optical Coherence Tomography (OCT) used to image mouse embryos before and after vitrification. Images A-C are unvitrified Metaphase I oocytes, 2PN and Metaphase II oocytes. Images D-F are images post-vitrification, where clumping of structures was observed and may be used to optimize the procedure. Identity of clumps is unknown (images courtesy of L. Zarnescu).

Figure 6.9 Representative image of phase subtraction microscopy of mouse embryos. Phase subtraction combines OQM and DIC microscopy and has been used to define blastomere boundaries and image embryos in the 3D plane, which may be used for cell counting. (images adapted from Warger et al. 2008).

Figure 6.10 Representative images of Qualitative Orientation Independent Microscopy (QOIM) of crane fly spermatocytes. QOIM uses DIC and polarization microscopy in tandem, and through the use of algorithms and computer-recombined images, produces a merged image (images adapted from Shribak et al. 2007, 2008).

Figure 6.11 Representative images of biodynamic imaging (BDI) of porcine (a) cumulus oocyte complexes and (b) blastocysts. Differences in cellular motions were detected between difference types of cells indicated a possible means of determining a non-invasive means of assessing subtle differences in cell quality (images adapted from An et al. 2015).

Chapter 7: Clinical Application of Methods to Select In Vitro Fertilized Embryos

Figure 7.1 Pre-implantation embryonic stages.

Chapter 9: The Non-Human Primate Model for Early Human Development

Figure 9.1 Induced DNA damage to sperm models, at least in part, human embryo developmental arrest. (a) control rhesus monkey ICSI embryo (no ROS treatment of sperm prior to fertilization), (b) ICSI embryo derived from ROS-treated motile sperm prior to fertilization of the oocyte, (c) phase contrast of normal four-cell ICSI rhesus embryo, (d) phase contrast of rhesus four-cell ICSI embryo displaying fragmentation of blastomeres, and (e) darkfield human embryo at the four-cell stage with fragments, a common arrest phenotype.

Figure 9.2 Rhesus embryo developmental landmarks for duration of cytokinesis in the first mitotic divisions after fertilization by intracytoplasmic sperm injection (ICSI).

Figure 9.3 Three rhesus blastocysts imaged using Eeva

TM

and one arrested embryo (C1).

Chapter 10: Cytoskeletal Functions, Defects, and Dysfunctions Affecting Human Fertilization and Embryo Development

Figure 10.1 (a–d) From left to right: (a) MII meiotic spindle in unfertilized human oocyte is oriented perpendicularly to the oocyte surface. Insets: enlarged meiotic spindle and enlarged oocyte centrosome without centrioles (acentriolar centrosomes) of one meiotic pole. (b) Sperm incorporation. Inset shows sperm centrioles before fertilization. After fertilization the sperm aster forms from the proximal centriole of the centriole pair while the distal centriole disintegrates. (c) Zygote aster formation and two apposed pronuclei. (d) Mitotic apparatus of first embryonic cell division. Inset: enlarged mitotic apparatus and enlarged centrosome of one mitotic pole containing centrioles.

Figure 10.2 (a) Schematic diagram depicting stages of preimplantation human development. The zygote forms during Day 1. At the 8-cell stage (Day 3), embryos start to undergo compaction; the blastomeres become flattened and polarized. After the 8-cell stage, the inner cells are formed (Day 5: 32 cell stage) resulting in the inner cell mass (ICM); the outer cells form trophectoderm (TE). Days 6 to 9: Blastocyst formation and continued cell differentiation.

Figure 10.2

List of Tables

Chapter 3: Molecular Biology of Endometriosis

Table 3.1 Seven important cellular pathophysiological processes integral to the development of endometriosis

Table 3.2 Genes in pathophysiology of endometriosis based on candidate gene studies

Table 3.3 Candidate genes in endometriosis based on association studies

Table 3.4 Genes showing anomalous expression in endometriosis revealed from studies using customised large scale array-based transcriptomic studies

Table 3.5 Genome-wide transcriptomic studies revealing genes with affected expression in endometriosis

Table 3.6 Summary of published studies on proteomic profiles in eutopic endometrium in endometriosis

Table 3.7 Regulatory functions of a few differentially expressed miRNAs in endometriosis.

1

Table 3.8 Seven major regulatory processes associated with endometriosis based on molecular biology evidence

Table 3.9 Future therapeutic targets in endometriosis

Chapter 5: Mitochondrial Manipulation for Infertility Treatment and Disease Prevention

Table 5.1 The implementation and outcomes of ooplasmic transfer

Chapter 6: Novel Imaging Techniques to Assess Gametes and Preimplantation Embryos

Table 6.1 List of novel imaging approaches as well potential applications or novel information gained from gametes and embryos

Chapter 7: Clinical Application of Methods to Select In Vitro Fertilized Embryos

Table 7.1 Key time-lapse studies evaluating prediction of blastocyst development

Table 7.2 Key time-lapse studies evaluating implantation/pregnancy

Table 7.3 Studies evaluating aneuploidy

Table 7.4 List of metabolites in the literature, which have been correlated to implantation in human embryos. The regulation symbols are as follows: ↑ or ↓ denotes if the consumption of a metabolite is increased or decreased, respectively, in implanted embryos compared to non-implanted embryos, ÷ denotes that no differences were detected

Table 7.5 List of proteins in the literature, which have been identified in conditioned culture media from human embryos. The regulation symbols are as follows: ↑ or ↓ denotes if a protein is increased or decreased compared to control samples, + or ÷ denotes if the protein was detected in a positive or negative implantation group. * indicates that the observed regulation was not significant

Chapter 8: New Horizons/Developments in Time-Lapse Morphokinetic Analysis of Mammalian Embryos

Table 8.1 Different types of TLM systems that have been used in human embryo research in the literature

Table 8.2 Commercially available TLM systems that are currently available and used in human ART Laboratories

Table 8.3 Recent TLM studies and their correlation status with certain embryonic stages/events

Human Reproduction

Updates and New Horizons

Edited by

Heide Schatten

Department of Veterinary Pathobiology,

University of Missouri, Columbia, Missouri, USA

Copyright © 2017 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Names: Schatten, Heide, editor.

Title: Human reproduction : updates and new horizons / edited by Heide Schatten.

Description: Hoboken, New Jersey : John Wiley & Sons Inc., [2017] | Includes index.

Identifiers: LCCN 2016038683| ISBN 9781118849583 (cloth) | ISBN 9781118849576 (epub)

Subjects: LCSH: Human reproduction.

Classification: LCC QP251 .H845 2017 | DDC 612.6–dc23 LC record available at https://lccn.loc.gov/2016038683

Cover image: Getty Images/mikroman6

List of Contributors

G. Anupa

Department of Physiology

All India Institute of Medical Sciences

New Delhi

India

Mustafa Bahceci

Bahceci Women's Health Group

Istanbul

Turkey

Muzaffer Ahmed Bhat

Department of Physiology

All India Institute of Medical Sciences

New Delhi

India

Antonin Bukovsky

The Laboratory of Reproductive Biology BIOCEV Institute of Biotechnology

Academy of Sciences of the Czech Republic

Prague

Czech Republic

Douglas T. Carrell

Andrology and IVF Laboratory

Department of Surgery (Urology)

Department of Obstetrics and Gynecology

and Department of Human Genetics

University of Utah

Salt Lake City

Utah

USA

Catherine M.H. Combelles

Biology Department

Middlebury College

Middlebury

Vermont

USA

Thomas F. Dyrlund

Department of Molecular Biology and Genetics

Aarhus University

Aarhus

Denmark

Necati Findikli

Bahceci Women's Health Group

Istanbul

Turkey

Debabrata Ghosh

Department of Physiology

All India Institute of Medical Sciences

New Delhi

India

Hans Jakob Ingerslev

The Fertility Clinic

Aarhus University Hospital

Aarhus

Denmark

Tetsuya Ishii

Office of Health and Safety

Hokkaido University

Hokkaido

Japan

Kirstine Kirkegaard

Department of Medical Biochemistry

Aarhus University Hospital

Aarhus

Denmark

Stuart Meyers

Department of Anatomy

Physiology

and Cell Biology

School of Veterinary Medicine

University of California

Davis

California

USA

Renee Riejo-Pera

Department of Cell Biology and Neurosciences

and Department of Chemistry and Biochemistry

Montana State University

Bozeman

Montana

USA

Heide Schatten

Department of Veterinary Pathobiology

University of Missouri

Columbia

Missouri

USA

Jayasree Sengupta

Department of Physiology

All India Institute of Medical Sciences

New Delhi

India

Munevver Serdaroğullari

Bahceci Women's Health Group

Istanbul

Turkey

Monis B. Shamsi

Andrology and IVF Laboratory

Department of Surgery (Urology)

University of Utah

Salt Lake City

Utah

USA

Luke Simon

Andrology and IVF Laboratory

Department of Surgery (Urology)

University of Utah

Salt Lake City

Utah

USA

Qing-Yuan Sun

State Key Laboratory of Reproductive Biology

Institute of Zoology

Chinese Academy of Sciences

Beijing

China

Jason E. Swain

Center for Reproductive Medicine

Department of Obstetrics & Gynecology

University of Michigan

Ann Arbor

Michigan

USA

Chapter 1Sperm Selection Techniques and their Relevance to ART

Luke Simon1, Monis B. Shamsi2 and Douglas T. Carrell1,2,3

1Andrology and IVF Laboratory, Department of Surgery (Urology), University of UT, Salt Lake City, UT, USA

2Department of Obstetrics and Gynecology, University of UT, Salt Lake City, UT, USA

3Department of Human Genetics, University of UT, Salt Lake City, UT, USA

1.1 Introduction

Fertilization is now possible using any available sperm through intra-cytoplasmic sperm injection (ICSI) treatment (Palermo et al., 1992). As a result, andrological research has raised questions regarding the selection of suboptimal sperm used for assisted reproductive technology (ART) (Avendano and Oehninger, 2011). In recent years, the role of sperm in ART has been highlighted as the sperm provides one half of the genome to the developing embryo. The use of healthier sperm has showed to improved ART outcomes and subsequently, sperm selection has become an integral part of ART procedure (Said and Land, 2011). Since the birth of first in vitro fertilization conceived baby in 1978, sperm selection for ART has been focused on selecting physiologically motile and morphologically normal sperm (Bartoov et al., 2002). Despite success, it has become evident that physical appearances of the sperm are inefficient to identify the most suitable sperm for ART success (Yetunde and Vasiliki, 2013). Hence, recent research is focused on developing novel sperm biomarkers to identify non-apoptotic sperm with high DNA integrity for successful use in ART.

Our understanding of sperm physiology, as well as the technology to select healthier sperm has progressively been improved. Initially, sperm selection was based on simple semen washing procedures and now more sophisticated sperm separation measures have evolved (Simon et al., 2015). The sperm is regarded unusable for the use in ART, after being analyzed for its molecular parameters such as DNA integrity, histone retention, protamine content, or ratio, and so on. Therefore, preserving the structural and functional integrity of the sperm is been the goal for recently introduced novel sperm selection approaches (Berkovitz et al., 2006a, 2006b). Some novel sperm selection approaches aim to mimic the natural selection process, where the female reproductive tract is known to eliminate poor quality sperm to enhance the chances of a successful fertilization (Holt and Fazeli, 2015). Other methods have focused on sperm physiological changes in the female reproductive tract, like capacitation, which are functionally important for acrosome reaction (Bedford, 1963). Inclusion of such novel biomarkers along with standard sperm preparation procedures has shown promises to enhanced fertilizing ability and improves ART success (Nasr-Esfahani et al., 2008a; Kheirollahi-Kouhestani et al., 2009; Polak de Fried and Denaday, 2010; Wilding et al., 2011).

1.2 Need of Sperm Selection in ART

Human semen is comprised of heterogeneous sperm population with varying degrees of structural differentiation, maturity, fertilizing ability, and functional quality (Huszar et al., 1993, 1998). During natural conception the sperm from these subpopulations compete to traverse through several anatomical and physiological barriers in the female tract. The most competent and reproductively efficient sperm are able to migrate through the cervical mucosa, uterus, uterine tube, cumulus cells, zona pellucida, and finally oolemma to participate in the fertilization (Suarez and Pacey, 2006). Further, selection takes place at the level of sperm-oocyte interaction and out of a population of millions, a single sperm is able to fertilize the oocyte and develop into an offspring. These barriers for natural selection exclude the sperm with structural abnormalities as acrosomal absence, flagellar deformity, immature sperm, and sperm with aneuploidy or other chromatin abnormalities from participating in a successful fertilization (Suarez and Pacey, 2006). On the contrary, during ART, sperm are brought in proximity to oocyte, outside the female body, where no such anatomical and physiological barriers exist. Depending upon the technique of ART, either the sperm fertilize the oocyte on their own as in IVF or the sperm are injected into oocyte for fertilization as in ICSI. During ART, sperm does not have to overcome any anatomical and physiological barriers present in the female reproductive tract, natural sperm selection are bypassed. Therefore, it is imperative to have an efficient artificial selection process that maximizes the probability of successful pregnancy and birth of a healthy offspring. Further, the sperm selection procedures also help to enrich the concentration of good quality sperm that increases the chance of ART success.

Sperm contribute half of the genome to the offspring. Therefore, selection of sperm with intact chromatin and free of chromosomal abnormalities is important for ART success. Studies indicate that even if the best quality sperm are used for ICSI, approximately, 55% of the selected sperm have normal DNA (Ramos et al., 2004). The primary objective of sperm selection approaches is to select good quality or healthier sperm. In addition, sperm selection approaches are designed to reduce the physiological and oxidative damage induced to the sperm during the selection process. With these perspectives in sight, recent developments in sperm selection approaches are focused on physiological properties or morphological characteristics or behavior in the electric field or basis on their fluid kinetic properties. This chapter discusses some of the novel sperm selection techniques that have been the focus of recent research and may have the ability to revolutionize ART by improving the success rate, even in patients with severely compromised sperm parameters.

1.3 Methodology of Sperm Selection

1.3.1 Intracytoplasmic Sperm Injection

Intracytoplasmic sperm injection (ICSI) is a very useful gamete micromanipulation technique for treating couples with severely compromised sperm parameters. Since its introduction in 1992, ICSI has revolutionized ART by providing hope to couples to achieve a pregnancy, who had few chances of a natural conception or by in vitro fertilization (IVF). The basic principle of ICSI is to manually select the best sperm on the basis of motility and/or morphology and to inject it into an oocyte. The premise for such gamete micromanipulation is that it enables a successful fertilization, when a sperm is unable to fertilize on its own. During this procedure, initial events of fertilization like acrosome reaction are bypassed and now fertilization is possible with any available sperm.

1.3.1.1 Methodology

The oocytes retrieved after ovarian hyper-stimulation is placed in a petri dish (specific for ART) in which they are fertilized with a sperm. The whole process is done with the help of a CCD attached microscope using a micromanipulator. The basic steps for ICSI manipulation are as follows: the oocytes retrieved after hyper-stimulation are held by a specialized holding pipette in a micromanipulator. The most visually normal sperm by virtue of its motility and morphology are picked by ICSI pipette. During this step, sperm are usually visualized at 400× magnification to increase the chances of detecting and eliminating any sperm with morphological abnormalities. The pipette containing sperm is then carefully inserted through the membrane of the oocyte, into the cytoplasm. A sperm is injected into the cytoplasm and the pipette is carefully removed. The oocytes are then incubated and checked for pronuclear appearance to confirm fertilization after 24 hours. After a successful fertilization, the embryos are cultured until cleavage stage (Day 3 embryo transfer) or until blastocyst stage (Day 5 embryo transfer) into the uterus.

1.3.1.2 Advantages and Limitations

ICSI is the most widely used ART, accounting to 70–80% of the cycles performed (Palermo et al., 2009). ICSI has assisted millions of infertile couples to conceive, even with severely compromised sperm parameters, as severe oligozoospermia, asthenozoospermia, or both in the male partner. This technique has dropped down the number of sperm required for fertilization from several thousand to a single viable sperm. In men with obstructive or non-obstructive azoospermia, where there are no sperm in an ejaculate, testicular-epididymal sperm extraction (TESE) combined with ICSI has made it possible to sire a child (Vloeberghs et al., 2015).

Although ICSI has fulfilled the dreams of parenthood for millions of infertile couples, but there are risks and concerns for the health of mother and the child associated with this technique. ICSI bypasses numerous physiological events of fertilization, which has always been a safety concern related to this technique. Many hazards are not specific to ICSI they are common to most of the ART. Two specific demerits of ICSI are: the injection of media into an oocyte along with the sperm and the bypass of natural selection process (Sánchez-Calabuig et al., 2014).

During natural conception, the sperm pass through different barriers with in the female reproductive tract, so that the most capable sperm, with normal morphology and vigorous motility can fertilize the oocyte (Barratt and Kirkman-Brown, 2006; Suarez and Pacey, 2006). Three levels of barriers effectively hinder the reach of an abnormal sperm to an oocyte; (1) the microenvironment of the female reproductive tract, (2) the sperm-oviduct interactions at the caudal isthmus, and (3) the sperm-zona pellucida interaction (Suarez and Pacey, 2006). ICSI bypasses these steps of natural selection to select the best sperm (observed by an embryologist), since it does not involve the sperm–oviduct interaction and other processes as zona pellucida binding-penetration. Further, the presence of acrosomal enzymes from the unreacted acrosome is introduced into the oocyte during ICSI, which may lead to an increased risk of vacuole formation (Morozumi and Yanagimachi, 2005; Morozumi et al., 2006).

During ICSI, the selection of sperm is based on the embryologist experience, which usually rely on the motility and morphology of the sperm. Since, these sperm parameters are not always reflective of sperm DNA integrity, chances of selecting a poor DNA quality sperm for fertilization in ICSI is not ruled out (Celik-Ozenci et al., 2004). Therefore, in ICSI there is a realistic possibility that a sperm with high DNA fragmentation or a aneuploid sperm can be selected for fertilization, which may ultimately lead to adverse consequences from failed fertilization and retarded embryo development to increased rates of miscarriage and diseases in the offspring.

In the post-natal life, children born after ART have been observed to have lower birth weights and higher peripheral fat, blood pressure, and fasting glucose concentrations than controls (Fauser et al., 2014). A meta-analysis of 19 publications selected by a quality score based on sample size and appropriateness of control group observed that major malformation rates ranged from 0 to 9.5% in IVF, 1.1 to 9.7% in ICSI, while 0 to 6.9% after natural conception, leading to a statistically significant overall odd ratio of 1.29 (Rimm et al., 2004). Further, it has been reported that 90–100% of the ART children with Beckwith–Wiedemann had imprinting defects, as compared to 40–50% of the spontaneously conceived children with Beckwith–Wiedemann (Manipalviratn et al., 2009). Similarly, 71% of the Angelman Syndrome cases in ART children were attributed to epigenetic defects as compared to 5% of the naturally conceived children with Angelman Syndrome (Manipalviratn et al., 2009).

1.3.1.3 Conclusion

ICSI sperm selection is an option for couples who have failed the standard IVF treatment and benefits men with severe male infertility. ICSI selected sperm is directly injected into the oocyte, which provides the best chance of fertilization in couples with fewer available oocytes for treatment. Despite advantages, the absence of natural sperm selection process may lead to an increased risk of miscarriage due to injection of any available sub optimal sperm, which subsequently increases the risk of health issues in ICSI born children. Research into the effects of ICSI sperm selection method is still on going, as this technique is extensive in use for less than two decades. However, ICSI sperm selection method does improve the odds of treating an infertile man, but it does by remove the key elements that often lead to male infertility.

1.3.2 Intracytoplasmic Morphologically Selected Sperm Injection

The introduction of ICSI as a method of insemination revolutionized the treatment of male infertility. With the widespread use of ICSI, contradictory findings were reported in many studies with regard to sperm selection based on morphology. Some studies demonstrate that sperm morphology according to strict criteria (Kruger et al., 1986, 1988) has controversial prognostic value in ICSI outcomes (Svalander et al., 1996; De Vos et al., 2003; French et al., 2010) and does not seem to influence embryo quality or development (De Vos et al., 2003; French et al., 2010). Therefore, need for more stringent sperm selection procedures were recommended to effectively improve ART outcome. As a major development in this direction introduced by Bartoov et al. (1994, 2001, 2002), who devised a method of unstained, real-time, high magnification (6600×) examination of sperm called “motile sperm organelle morphology examination” (MSOME). The integration of MSOME with ICSI sperm selection was defined as intracytoplasmic morphologically selected sperm (IMSI) (Bartoov et al., 2003).

During IMSI, the motile sperm morphology that includes normalcy of the sperm nucleus (shape and chromatin content), acrosome size, presence and absence of vacuoles, are observed at high (6600×) magnification instead of around 400× used during conventional ICSI. The introduction of IMSI facilitated the observation of ultra-structural morphological details of live sperm, thereby assisting in selection of healthier sperm, to be used for ART.

1.3.2.1 Methodology

IMSI is a modification of ICSI, in which the sperm selection is done at magnification many fold higher than ICSI. Its introduction in the field of ART facilitated the observation of live human sperm, particularly by showing sperm vacuoles not necessarily seen at lower magnification. The sperm selection for IMSI relies on the evaluation criterion of MSOME, which evaluates the presence, size, number, and location of vacuoles. According to the MSOME criterion, if the sperm head contains one or more vacuoles (diameter of 0.78 ± 0.18 µm) occupying more than 4% of the normal nuclear area, it is considered abnormal for use in ART (Bartoov et al. 1994, 2001, 2002). The MSOME criterion has been modified to a scoring system, to simplify the sperm classification into different grades. Briefly, grade I sperm have normal sperm head and absence of vacuoles and they represent the optimal type. Grade II sperm are characterized by maximum two small vacuoles. Grade III sperm have either more than two small vacuoles or one large vacuole. The grade IV represents the poorest quality sperm, which show large vacuoles fully occupying the head, along with other morphological defects (Vanderzwalmen et al., 2008; Greco et al., 2013). Cassuto et al. (2009) introduced a similar protocol of sperm classification based on the detailed analysis of head, acrosome, vacuoles, base of sperm head, and the presence of cytoplasmic droplet.

1.3.2.2 Advantages and Limitations

The use of IMSI over ICSI or other sperm selection techniques has significantly improved ART success rate, since it involves the selection of sperm with a strictly defined, morphologically normal nucleus. It has been particularly useful for couples with repeated ICSI failure (Bartoov et al., 2003; Berkovitz et al., 2005; Hazout et al., 2006; Antinori et al., 2008; Franco et al., 2008; Setti et al., 2010). It has been reported that IMSI is associated with significantly higher implantation and clinical pregnancy rates and a reduction in the abortion rates (Setti et al. 2010, 2011), where the pregnancy rate in IMSI has been observed to be 66% as compared to 30% in ICSI. The reported implantation rate in IMSI is 27.9% while it is 9.5% in ICSI (Bartoov et al., 2003; Berkovitz et al., 2005). In cases, where no sperm could qualify for selection in the IMSI procedure, an increase in abortion rate from 10 to 57% has been reported (Berkovitz et al., 2005). Further, Cassuto et al. (2014) reported a lowering of congenital malformation in IMSI born children to 1.3% as compared to 3.8% born after ICSI. In addition, IMSI improved ART outcome in patients with severe degrees of sperm DNA damage. It has also provided evidence for the association of presence, size, and number of sperm nuclear vacuoles with embryo quality and development, and suggested that high number of vacuoles may account for increased abortions (Figueira et al., 2011).

Though, IMSI has been documented to significantly improve the ART outcomes, but the technique has its drawbacks. Undoubtedly, it is a time-consuming technique and selecting a normal sperm in accordance with MSOME criterion may take 60–120 min (Antinori et al., 2008). Further, the prolonged exposure to the microscope's heated stage may itself cause damage to the sperm, as demonstrated by Peer et al. (2007) after 2 h on the microscope's heated stage, sperm nucleus vacuolization significantly increases. Despite these observations, IMSI has proved itself as a valid tool for safe and a non-invasive method of sperm selection.

1.3.2.3 Conclusion

The IMSI sperm selection approach changed the perception of how a sperm suitable for insemination should appear. Sperm, which was considered as normal when observed under a low magnification microscope, is showed to contain ultra-structural defects that may impair ART outcomes. Recent studies have reported that IMSI is associated with improved ART outcomes; specifically, implantation and pregnancy rates, while a reduction in miscarriage rates was observed when compared to conventional ICSI insemination. Despite its advantages, clinical indications for IMSI procedure are still lacking and further prospective randomized clinical trials are required to identify patient groups that are benefited by IMSI sperm selection approach.

Figure 1.1 Apoptotic sperm are labeled by annexin V magnetic beads. A magnetic field separates the apoptotic sperm.

1.3.3 Annexin V Labeling

Annexin V labeling is a well-recognized method to detect bio-molecules at the sperm membrane to identify apoptotic sperm. This method has been widely used to separate the apoptotic sperm from non-apoptotic (healthier) sperm population. This method is based on the affinity of protein coagulant, Annexin V, with a phospholipid, phosphatidylserine of sperm plasma membrane. In a normal sperm, phosphatidylcholine and phosphatidylserine are asymmetrically distributed, with the former exposed to external leaflet of membrane while the later located at the inner surface of lipid bilayer. However, this asymmetry is disrupted during apoptosis, when the phosphatidylserine is externalized to the outer side of membrane, which facilitates an apoptotic sperm to be recognized by the macrophages and eliminated. A magnetic bead-conjugated annexin V helps in the identification of an apoptotic sperm, in an external magnetic field, annexin-V conjugated to dead and apoptotic sperm by magnetic activated cell sorting (MACS; Figure 1.1).

1.3.3.1 Methodology

Annexin V is a phospholipid binding protein that has high affinity for phosphatidylserine but lacks the ability to pass through an intact sperm membrane (van Heerde et al, 1995). Therefore, in sperm with compromised membrane integrity, the annexin V binding takes place at the phosphatidylserine exposed on the outer layer of membrane (Glander and Schaller, 1999). To separate the apoptotic sperm from non-apoptotic sperm, super-paramagnetic microbeads conjugated with annexin V are used to label sperm with externalized phosphatidylserine. During this procedure of MACS, a mixture of sample and conjugated annexin V is incubated, and loaded on a separation column placed in the magnetic field. The attractive force between the magnetic field around the column attracts the magnetic beads conjugated to annexin V-sperm complex, and hence the annexin V-positive fraction comprising of apoptotic sperm binds to the column, while the annexin V-negative fraction of non-apoptotic sperm elutes through the column. The column is removed from the magnetic field, and annexin V-positive fraction is eluted using the annexin V-binding buffer (Chan et al., 2006). Thus, this method yields two fractions: annexin-negative (intact membranes, non-apoptotic sperm) and annexin-positive (externalized phosphatidylserine, apoptotic sperm) (Grunewald et al., 2001; Glander et al, 2002).

1.3.3.2 Advantages and Limitations

Annexin V labeling is a simple, convenient method for detection and separation of apoptotic sperm. It provides optimal purity with reliable and consistent results. As opposed to other routine methods of sperm separation, which rely on motility and sperm density, this technique acts at the molecular level. Combining this method, with other techniques such as density gradient centrifugation may yield a sperm population with higher motility, viability, and lower number of apoptotic sperm, though it makes the procedure for sperm isolation more time and energy consuming (Said et al., 2006a). This technique has been reported to improve acrosome reaction and is associated with higher cleavage and pregnancy rates than spermatozoa selected by density gradient centrifugation in oligoasthenozoospermic men (Dirician et al., 2008; Lee et al., 2010). Annexin V negative fraction has low amounts of DNA damage, and higher oocyte penetration capacity than annexin V-positive sperm (Said et al., 2006). Although, sperm sorting with annexin V method was effective in the treatment couples with previously failed ICSI outcome (Polak de Fried and Denaday, 2010; Rawe et al., 2010), Romany et al. (2014) reported no improvement in ART outcomes when comparing MACS technology to remove apoptotic sperm with swim-up method.

An important limitation of this method is that annexin V may bind with non-apoptotic cells having damaged plasma membrane with the exposed phosphatidylserine and may exaggerate the percentage of apoptotic cells. Secondly, it has been reported that live and healthy macrophages or monocytes, after ingestion of apoptotic bodies or fragments of apoptotic cells become annexin V positive and thus may be misidentified as apoptotic cells (van Engeland et al., 1998). The effect of using magnetic beads in ART has raised concerns that these foreign particles may be accidentally injected to the oocyte along with normal sperm, however this method has shown promise in some trials (Polak de Fried and Denaday, 2010; Rawe et al., 2010), but this technology is yet to be tested in larger randomized trials.

1.3.3.3 Conclusion

Annexin V-conjugated magnetic beads can separate sperm with externalized phosphatidylserine, which is considered one of the early features of late apoptosis. Removal of sperm, which failed to be excluded by the apoptotic machinery or with abnormal membrane protein, should theoretically benefit sperm selection. The separation of non-apoptotic sperm with intact membranes may enhance cryosurvival rates following cryopreservation (Said et al., 2005). Although, this method can effectively remove apoptotic sperm, however there are other components in semen such as leukocytes, debris, and so on that should be removed. Therefore, integration of MACS with density gradient centrifugation can be considered as an effective approach to select non-apoptotic perm (Said et al., 2006b).

1.3.4 Microfluidics