Beauty lies in the eye of the beholder

The connection of beauty to the eyes of the beholder is much deeper than what it looks. Different people have different perception of reality or on things. Some might find somethings good while other may find it bad. In this study let us concentrate on Morphological inspection.It is the best tool and it has wide range of observations for a given sample…Let’s find out how?. The image below was asked for assessment to the embryologists in a conference and was told to grade the oocyte accordingly.

Oocyte image given for the quiz [courtesy : ESHRE Atlas of human embryology]

 

Around 20 of them took up the quick quiz. The image that was given during the quiz was taken from ESHRE Atlas of human embryology. Below the image were a list of buttons which were related to the grading of the oocytes like: Maturity, Size, Zona, SER etc. They had to select the appropriate options referring to the image.

The result of the quiz was made into a pie charts for better representation and understanding. We were surprised to see the answers obtained.

Zona appearance, for example appeared to be Uniform for 63.2% of them and Non-uniform for 36.8%.

Likewise, maturity of oocyte for a M2 oocyte was 83.3%, M1- 11.1% and GV 5.6%.

Maturity graph [M2;GV;M1]

Grading the oocyte based on quality, 50% has told it is good, 25% abnormal, 15% approximately mature and 10% said almost normal.

Grade quality of the oocyte

The polar body had sections like – Shape, texture, size and fragments. Shape: 2 of them found it to be irregular, 10 of them opted oval and 5 of them found the oocyte to be round. Based on the texture of PB- 10 of them found it smooth and 7 of them rough. The PB size was selected normal by 15 of them, smaller and enlarged by 5 of them. 7 of them found the PB to be fragmented whereas 9 of them found the PB to be normal without any fragments.

Polar body grading

 

Zona pellucida grading of the oocyte

Vacuoles and SER have got the same percentage of answers. Absent is 62.5% and Present is 37.5%.

Vacuole and SER grading of the oocyte

 

Bio-Insights- Sperms

Structure of Spermatozoon 

A morphologically normal sperm cell is about 40-50 µm in length and consists of a head and tail. [2]

Head: It mainly consists of nucleus and acrosome. Sperm head performs two functions- genetic and activation. The genetic function is embodied in the sperm nucleus which consists of DNA and nuclear proteins and thus is responsible for the transmission of hereditary characters from the male. The major part of the sperm head is occupied by the nucleus about 65%, which determines the sperm head shape. The sperm head anterior end is covered by a cap-like structure called acrosome. The acrosome is represented by Golgi complex and it contains a number of hydrolytic enzymes, such as hyaluronidase and acrosin, which are required for fertilization. During fertilization, the acrosomal membrane fuses with the oocyte cytoplasmic membrane and followed by acrosomal reaction, an event where the acrosomal enzyme is released from the head tip. Sperm head measures between 4.0-5.5 µm in length and 2.5-3.5 µm in width. [1 and 2]

Structure of a Sperm

Neck: It is a short, slightly constricted segment made up of projections located between the head and the tail portion. Neck differs clearly from the head and also from the rest part of the tail. [1 and 2]

Tail: The tail measures 40-50 µm in length and provides motility for the cell. Sperm cell’s entire motility apparatus is contained in the tail. The tail can be divided into the mid-piece (anterior portion), principle piece, and end-piece (posterior portion). Mid-piece supports the head at exactly the center position. The mid-piece consists of tightly packed mitochondria surrounded by a sheath. The mitochondria in the mid-piece supply energy in the form of ATP for tail movement. The principle piece is the longest part of the tail and comprises most of the propellant machinery. Motility plays the main role in sperm transport through the cervix. [2]

 

Abnormal Sperm Morphology

Teratospermia is a condition characterized by the presence of sperm with an abnormal morphology that affects fertility in males.  Normal sperm exhibits an oval-shaped head with a regular outline and a cap (acrosome) covering more than one-third of the head surface. The mid-piece is slender, less than one-third of the width of the head, straight and regular in outline. The tail is slender, uncoiled and should present a regular outline. Abnormal sperm morphology is classified as a defect in the head, midpiece or tail of the sperm.

Sperm Head Abnormalities

Head defects include large, small, tapered, pyriform, round, amorphous heads, heads with a small cap area and double heads, as well as any combination of these. Globozoospermia, where the sperm head appears small and round due to the failure of the acrosome to develop is one of the examples of a head defect.

Mid-piece defects include “bent” neck (where the neck and tail form an angle greater than 90% to the long axis of the head), thick/irregular mid-piece, abnormal thin mid-piece, as well as any combination of these.

Sperm Tail Abnormalities

Tail defects Include short, multiple, hairpin, broken or bent (>900) tails, tails of irregular width, coiled tails, as well as any combination of these.

 

Sperm Defect

Possible related observations Possible associated functional anomaly

Elongated head

Abnormally shaped head and abnormally condensed chromatin Immature chromatin/fragmented DNA/increased aneuploidy

Thin head

Abnormally shaped head and abnormally condensed chromatin

Immature chromatin/fragmented DNA

Microcephalous head

Excessive shrinkage of the nucleus and abnormally condensed chromatin

Immature chromatin/fragmented DNA

Macrocephalous head

Insufficient shrinkage of the nucleus and abnormally condensed chromatin

Increased aneuploidy

Multiple heads

Two or more closed or dissociated heads with or without a common acrosome or midpiece

Handicaps migration through mucus and oocyte vestments/fragmented DNA

Abnormal acrosome region

Absent or abnormally shaped or sized acrosome, incomplete acrosome and/or abnormal appearance of the underlying nucleus

Abnormal acrosome reaction/fragmented DNA

Thin midpiece

Partial/absent mitochondrial sheath

No or reduced ATP available for cell propelling

Bent tail

Misaligned midpiece and head or sharply bent midpiece/tail

Impairment of syngamy and cleavage, abnormal cell propelling, handicaps migration through mucus and oocyte vestments

Absent tail

Various anomalies of the neck region

Fragility of the neck structure and sperm moving forward is not possible

Short tail

Abnormally shaped periaxonemal and sometimes axonemal structures/dysplasia of the fibrous sheath

Immotility or severe dyskinesia

 

Irregularly shaped tail

 

Abnormally shaped periaxoneamal and sometimes axonemal structures/dysplasia of the fibrous sheath

Abnormal motion

Coiled tail

Completely or partially coiled tail often within a huge cytoplasmic remnant

Sperm moving forward is not possible

Multiple tails more than one tail

Partially dissociated tails connected to a single or to multiple heads or tails knitted together over a variable length

Abnormal motion: handicaps migration through mucus oocyte vestments

 

Semen Analysis

This is an evaluation of a certain characteristic of male’s semen and the sperm. This helps in evaluating male fertility

  • Color: Normally semen appears in whitish-gray in color. As the man ages, it appears as a yellowish tint. Presence of blood in semen (hematospermia) a rare condition which results in brownish or red color ejaculate. Deep yellow or greenish color appearance of semen is due to medication. Other causes of unusual semen color are due to STI’s, genital surgery and injury to male sex organs.
  • Volume: Semen volumes between 2.0 ml and 5.0 ml are considered to be normal. WHO regards 1.5 ml as the lower reference limit.
  • pH: According to WHO the normal semen pH is in the range of 7.2-8.2. An acidic pH ejaculate indicates one or both of the seminal vesicles are blocked. A basic pH ejaculate indicates an infection. pH value outside of the normal range is harmful to sperm and affects their ability to penetrate the egg.
  • Viscosity: It measures seminal fluid’s resistance to flow. High viscosity may interfere with the determination of sperm motility, concentration and antibody of spermatozoa. Normally semen coagulates upon ejaculation and usually liquefies within 15-20 min. Liquefaction time within 60 min is considered as the normal range.
  • Motility: The efficient passage of spermatozoa through the cervical mucus is dependent on rapid progressive motility, that is, spermatozoa with a forward progression of at least 25 µm/s. Reduced sperm motility can be a symptom of a disorder related to male accessory sex gland secretion.

>Rapid progressive motility- Moves at >25 µm/s at 370C and >20 µm/s at 200C

>Non-progressive motility- Move at <5 µm/s

>Immotility

Reference:

  1. http://www.biologydiscussion.com/notes/structure-functions-and-types-of-mature-sperm-in-animals-biology/768
  2. https://www.researchgate.net/publication/283864069_Sperm_Biology_from_Production_to_Ejaculation
  3. file:///C:/Users/BBC_common/Downloads/fertilitypedia-abnormal-sperm-morphology%20(1).pdf
  4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3114587/
  5. https://en.wikipedia.org/wiki/Semen_analysis

 

 

Bio-Insights-Embryo

Embryo Development

The product of fertilization is an embryo. The first stage is the one-cell embryo with a diploid number of chromosomes. The embryo undergoes a series of cell divisions, for few days to form a hollow sphere of cells known as blastocyst. [1]

Day-1, 2PN stage, in this stage, the embryo contains two polar bodies and two pronuclei with small nucleoli. One of the pronuclei contains genetic information from the egg and the other from the sperm. The pronuclei are usually checked between 16-18 hours after the sperm injection. [2]

The one cell embryo undergoes a series of cleavage divisions, progressing through 2-cell, 4-cell, 8-cell and 16-cell stages. The cells in cleavage stage embryos are known as blastomeres. Early on, cleavage divisions occur quite synchronously. In other words, both blastomeres in a two-cell undergo mitosis and cytokinesis almost simultaneously. Hence, due to these reasons, the embryos are most commonly observed at two, four and eight-cell stages. Embryos with an odd number of cells (e.g. 3,5,7) are less commonly observed, simply because those states last for a relatively short time. [1]

After the development of 8-cell embryo, the blastomere begins to form tight junctions with one another, leading to deformation of their round shape and formation of a mulberry-shaped mass of cells called a morula. This change in shape of the embryo is called compaction. It is difficult to count the cells in a morula, the embryo shown here probably has between 16 to 32 cells. [1 and 3]

Junctional complexes formed between the blastomeres gives the embryo outside and inside. The outer cells of the embryo express variety of membrane transport molecules, including sodium pumps, which results in an accumulation of fluid inside the embryo, which signals the formation of the blastocyst.

A blastocyst is composed of a hollow sphere of trophoblast cells, inside of which is a small cluster of cells called the inner cell mass. Trophoblast goes on to contribute to fetal membrane systems, while the inner cell mass is destined largely to become the embryo and fetus. Eventually, the stretched zona pellucida develops a crack and the blastocyst escapes by a process called hatching. This leaves an empty zona pellucida and a zona-free or hatched blastocyst lying in the lumen of the uterus. [1]

Embryo assessment

The most important morphological parameters to asses in the laboratory are blastomere appearance, fragmentation, and multinucleation. Those with equal blastomeres, minimal cytoplasmic fragmentation, and few multinucleated cells show a better prospect of implantation. [4]

  • Blastomere size

The relative blastomere size in the embryo is dependent on both the cleavage stage and the regularity of each cleavage division. [3]

  • Fragmentation

The fragmentation of the cytoplasm is therefore defined as the presence of anucleate structures of blastomeric origins. The degree of fragmentation is most often expressed as the percentage of the total cytoplasmic volume. The relative degree of fragmentation is defined as mild (<10%), moderate (10-25%) and severe (>25%). High degree of fragmentation correlates negatively with implantation and pregnancy rates, while the presence of minor amounts of fragmentation has no negative impact. [3]

  • Nucleation

The nucleation status is defined as the presence or absence of nuclei in the blastomeres of the cleavage stage embryo. Nucleation status of each blastomere in the embryo is evaluated as a single nucleus per blastomere, no nuclei visible or multinucleation. [3]. Multinucleation arises due to karyokinesis in the absence of cytokinesis with subsequent arrest of the blastomere or the entire embryo. Multinucleation may also be caused by partial fragmentation of nuclei or by defective migration at mitotic anaphase. [4] Multinucleation is predictive of a decreased implantation potential and are associated with an increased level of chromosomal abnormalities and increased risk of spontaneous abortion.

 

2PN embryo

The human oocyte arrested in the second meiotic division (M-II), usually characterized by the presence of a first polar body. The entry or injection of the sperm into the oocyte completes the second meiotic division. The second polar body containing the chromatids from one haploid chromosome set is extruded, and the female pronucleus is formed. During this process, the ooplasm rotates in a periodical way and in parallel the sperm chromatin decondenses. The sperm cell also delivers the centriole that has a leading role in further development and control of microtubules that are important for the symmetry of the developing embryo. These microtubules pull the haploid female pronucleus towards the male pronucleus. Both pronuclei finally migrate to the center of the cell and align. The G1 phase starts approximately 2-3 hr after sperm entry and pronuclei appear after 4-6 hr. The process is complete 18-22 hr after sperm entry or injection. [5]

 

Cleavage stage embryos

Embryos in cleavage stage range from the 2-cell stage to the compacted morula composed of 8-16 cells. Good quality embryos must exhibit appropriate kinetics and synchrony of division. In normal-developing embryos, cell division occurs every 18-20 h. Embryos diving either too slow or too fast may have metabolic and/or chromosomal defects. The blastomeres dividing in exact synchrony produces only 2, 4 or 8 cell embryos. Asynchronous developments lead to the formation of 3, 5, 6, 7 or 9 cell embryos. [3]

Mitosis in blastomeres should produce two equally sized daughter cells. When the division is asymmetric, one of the blastomeres of the next generation will inherit less than the amount of cytoplasm of parent blastomere, leading to a defective lineage in the embryo. After two cleavages, the zygote becomes a 4-cell embryo, where the 4-cells are normally arranged in a tetrahedron in the spherical space provided by the ZP. Blastomeres located close to a single, spatial plane produced by an incorrect orientation of division axes will lead to altered embryo polarity. [3]

After the embryo reaches the 8-cell stage, the blastomeres begin to show an increase in cell-cell adherence due to the spread of intercellular junctions. This is the start of compaction. The process of compaction advances during the next division until the boundaries between the cells are barely detectable. If some of the blastomeres are excluded from this compaction process, the embryo may have a reduced potential for becoming a normal blastocyst. [3]

 

Blastocyst

The blastocyst consists of cells forming an outer trophectoderm (TE/trophoblast) layer, an inner cell mass (ICM, embryoblast) and a blastocoel (fluid-filled cavity). The ICM forms an inner layer of larger cells also called as “embryoblast” which are the cluster of cells located and attached on one wall of the outer trophoblast layer. In week 2 of embryo development, ICM differentiates into two distinct layers the epiblast and hypoblast. ICM is the source of true embryonic stem cells capable of forming all cell types within the embryo. [7]

The trophectoderm (TE) outer layer of smaller cells is also called the “trophoblast” epithelium. The key function is for the transport of sodium (Na+) and chloride (Cl-) ions through this layer into the blastocoel. In week 2 this layer will differentiate into two distinct trophoblast layers the syncytiotrophoblast and cytotrophoblast cells and are key to implantation and early placentation. [7]

 

Blastocyst Hatching

As the fluid and number of cells inside the blastocyst increase its progressively causes enlargement of the blastocyst and its cavity with a consequent progressive thinning of the zona pellucida (ZP). Finally, the blastocyst breaks free of the ZP through a process called hatching.

 

Cytoplasmic Anomalies

The cytoplasm of cleaving embryos is normally pale, and clear or finely granular in appearance. Cytoplasmic anomalies, such as cytoplasmic granularity, cytoplasmic pitting and the presence of vacuoles, occur occasionally.

Cytoplasmic pitting is characterized by the presence of numerous small pits with an approximate diameter of 1.5 µm on the surface of the cytoplasm. The cytoplasm of blastomeres may be excessively darkened with centralized granularity, where these kinds of embryos have reduced implantation potential or can undergo degeneration.

Cytoplasmic vacuolization varies in size and number. Vacuoles are membrane-bound cytoplasmic inclusions filled with fluid that are virtually identical with the perivitelline fluid. Extensive vacuolization is always considered as detrimental. [6]

  1. http://www.vivo.colostate.edu/hbooks/pathphys/reprod/fert/cleavage.html
  2. https://ivf.net/ivf/embryo-development-o2591.html
  3. http://atlas.eshre.eu/es/14611830864735920
  4. The Infertility Manual, Editor: Kamini A Rao and Co-editor: Howard Carp
  5. https://books.google.co.in/books?id=Kp5_AwAAQBAJ&pg=PA111&lpg=PA111&dq=vacuoles+in+embryo+eshre&source=bl&ots=VCxpH1yiuz&sig=t1ARQo-CeYN24Isgl18iSY-3qeg&hl=en&sa=X&ved=2ahUKEwjBmqzNmsvcAhWCbn0KHTFfD3I4ChDoATABegQIAhAB#v=onepage&q&f=true (Morphological selection of gametes and embryos: 2PN/zygote by Martin Greuner and Markus Montag)
  6. http://atlas.eshre.eu/es/14611418225805670
  7. https://embryology.med.unsw.edu.au/embryology/index.php/Blastocyst_Development

Embryo Biopsy

  1. Blastomere Biopsy

Blastomere biopsy is a technique that is performed by removal of one or two cells (blastomeres) from 4-8 cell embryo for preimplantation analysis. On the third day of the embryo development, the embryo is maintained in a position by a pipette with rounded margins, an opening is made in the embryo by using a laser device or treating it with thyroid acid. Once the hole is made the cells from the embryo are removed using a micropipette having a greater diameter. At this stage of embryo development, all the cells are equivalent and thus removal of a cell from the embryo at this stage does not remove anything critical for normal development. The embryo compensates for the removed cell and should continue to divide following blastomere biopsy.  After removal of cells from the blastomere, the developing embryo is placed back into the culture dish and the removed cells are used for subsequent genetic analysis. [1]

2.  Trophectoderm Biopsy

Trophoblast/Trophectoderm are cells forming the outer layer of a blastocyst, which provide nutrient to the embryo and develop into a large part of the placenta. They are formed during the first stage of pregnancy and are the first cells to differentiate from the fertilized egg. [3] Trophectoderm biopsy involves removing some cells from the trophectoderm component of a blastocyst embryo at day 5/6.

Reference:

  1. http://www.preimplantationgeneticdiagnosis.it/polar-body-removal-blastomere-biopsy.htm
  2. https://nordicalagos.org/trophectoderm-biopsy/
  3. https://en.wikipedia.org/wiki/Trophoblast

 

Preimplantation Genetic Screening (PGS) and Preimplantation Genetic Diagnosis (PGD)

PGD: It was first introduced in the year 1989. A procedure to test the embryos for specific conditions before implantation in couples who are at risk of transmitting genetic abnormality to their offspring. The embryos are biopsied at either zygote, cleavage or blastocyst stage. Used to determine embryo genotype, performed in couples with genetic abnormalities such as single-gene disease, single mutations, translocations or other gene abnormalities. The embryos can be biopsied at different stages like zygote stage (removal of the first and second polar body), cleavage stage (removal of one to two blastomeres from the six to eight cell embryo) and blastocyst stage (removal of some trophectoderm cells). Almost all PGD cycles are carried out during blastomeres after the cleavage-stage biopsy. Polar body biopsy is rarely used as it only gives genetic information on the maternal genome. [1]

Testing by Fluorescence in-situ hybridization (FISH)

FISH involves identification of chromosomes or their fragments with fluorescently labeled molecular probes. Probes are complementary to specific DNA regions that are subject to hybridization under specific conditions, and the result of this process can be observed as fluorescent spots under a fluorescent microscope. In FISH it is not possible to test a whole panel of 24 chromosomes during one test, as it is only feasible to use 5-9 probes at most, for 2-3 rounds of hybridization. Hence, it is limited to the most common abnormalities involving chromosomes 13, 15-18, 21, 22, X and Y. Due to a lot of disadvantages of FISH, this field of study has now been successfully replaced by methods that are more reliable and precise, such as SNP and NGS. [2] This technique is usually referred to as preimplantation genetic screening (PGS) and should be differentiated from PGD, as it is for a different group of patients and for a different reason. [1]

Testing by PCR

For couples at risk of single gene disorder, PGD is carried out using PCR. [1]

Whole genome approaches

The introduction of whole genome amplification (WGA) methods has enabled high throughput technologies to be used, which has increased the amount and type of information that can be obtained from an embryo biopsy sample. Techniques that come under WGA are preimplantation haplotyping (PGH), Array comparative genome hybridization (CGH), Next-generation sequencing (NGS). These whole-genome approaches rely on whole-genome amplification. [1]

PGS: this is used to determine potential aneuploidies of all 24 chromosomes. Particularly performed for patients older than 35 (advanced maternal age), those with repeated implantation failures, repeated miscarriages, with severe male factor infertility.

Reference:

  1. https://www.rbmojournal.com/article/S1472-6483(15)00605-7/pdf
  2. https://www.researchgate.net/publication/306004985_Current_Methods_for_Preimplantation_Genetic_Diagnosis

 

Bio-Insights- Oocyte Morphological features

Smooth Endoplasmic Reticulum (SER) role in oocyte

SER’s are translucent vacuoles observed occasionally during ICSI in the cytoplasm of the egg. [2] testing

The endoplasmic reticulum (ER) is a network of membranes found throughout the cell and connected to the nucleus. ER functions as a manufacturing and packaging system to make products such as hormones and lipids. [2]

There are two types of ER, Rough ER, and Smooth ER

SER’s are easily distinguishable from fluid-filled vacuoles because they are not separated from the rest of the ooplasmic volume by a membrane, and are seen as translucent vacuoles. [3]

SER helps in steroid hormones and fat metabolism and production. It is smooth due to association with smooth slippery fats and is not studded with ribosomes. SER ‘s pivotal role is to store and release calcium, which will affect the calcium balance in SER-positive oocytes. .[2]

The mechanism of formation of SER’s are due to some functional and structural alterations of the SER during oocyte maturation, such as the increase in the sensitivity of the IP3 receptor for calcium, increase storage of calcium that is released during oscillation. In human oocytes, the localization of mobilizable calcium ions was detected in the small vesicles beneath the plasma membrane of SER. [3]

According to transmission electron microscopic analysis, there are three forms of SER’s, large (18 µM); medium (10-17 µM) which can be classified by light microscopy and small (2-9 µM) which are not visible under clinical embryology laboratory conditions. [3]

Oocytes consist of SER due to the presence of high estradiol level. In many cases, serum estradiol levels on the day of hCG administration were significantly higher in SER-positive cycles. [2]

It has been observed that the occurrence of SER’s is significantly related with longer duration and higher dosage of the stimulation. [3]

Pregnancies in women with affected gametes were accompanied by a higher obstetric problem leading to non-significant trends towards earlier delivery and significantly reduced birth weights. It is strongly recommended to avoid the transfer of embryos/blastocysts derived from SER-cluster positive gametes. It is known that even transfer of sibling oocytes without these anomalies involves high risk and detrimental outcome.

It is showed that the results of transfer of SER-positive embryos results in a high rate of miscarriage and the women tend to deliver earlier at 36.4 weeks of gestation which leads to lower birth rate 

References:

  1. https://www.rbmojournal.com/article/S1472-6483(10)60563-9/pdf
  2. https://www.slideshare.net/malpani/eggs-showing-smooth-endoplasmic-reticulum-clusters-produce-outcomes-similar-to-normal-eggs
  3. https://books.google.co.in/books?id=Kp5_AwAAQBAJ&pg=PA83&lpg=PA83&dq=refractile+bodies+in+oocyte&source=bl&ots=VCxnK7vkwu&sig=Akh6mi0lSgdMs36GtIulV5Zrgfo&hl=en&sa=X&ved=0ahUKEwi5mrDgspvcAhXIT30KHZkqAtI4ChDoAQhMMAc#v=onepage&q=refractile%20bodies%20in%20oocyte&f=false

Vacuoles role in oocyte

One of the most common oocyte dysmorphism is cytoplasmic vacuolization. Vacuoles are membrane-bound cytoplasmic inclusions filled with fluid that is virtually identical with perivitelline fluid. They vary in size as well as in number. They arise spontaneously or by fusion of preexisting vesicles derived from Golgi apparatus/SER. [1]

It has been shown that vacuolized oocytes have significantly reduced fertilization rates and developmental ability. Vacuoles of size 5-10 µM in diameter don’t show any biological consequences. A vacuole >14 µM in diameter can completely block fertilization. Single or multiple large vacuoles may displace the meiotic spindle from its polar position or disturb the cytoskeleton resulting in fertilization failure. [1 and 2]

Two types of the vacuole in oocytoplasm are seen. Type 1 vacuole is related to apoptosis. The formation of type 1 vacuoles is one of the morphological characteristics of apoptosis. The mechanism responsible for type 2 vacuoles is unknown. Type 1 vacuole can be seen very clearly and they look like lunar craters. Type 2 vacuoles are not obvious as type 1 vacuoles and are flat and more like a bulge than a crater. Type 2 vacuoles are common in MII oocytes. [1] 

References:

  1. https://www.rbmojournal.com/article/S1472-6483(11)00349-X/fulltext
  2. https://www.researchgate.net/publication/229326639_The_oocyte?_sg=hXmowSf9g-nhMoxNcXfHz3khl6F7c6fBTyrlIkTAeAcVyFJR1kzxL4RsmxYM_31frJPpJ8sq8w

Refractile bodies role in oocyte

Refractile bodies are cytoplasmic inclusions that can be dark incorporations, fragments, spots, dense granules, lipid droplets, and lipofuscin. TEM studies and Schmorl staining have shown the refractile bodies >5 µM in diameter showed the conventional morphology of lipofuscin inclusions that consisted of a mixture of lipids and dense materials. Lipofuscin bodies in human oocytes can be detected throughout meiotic maturation (GV, MI, and MII). Accumulation of lipofuscin occurs during the growth phase of the oocyte when dominant follicles are being recruited into the preovulatory pathway. The occurrence of large lipofuscin bodies in normal aging may also be related to conditions of the developing ovarian follicles, such as perifollicular blood circulation and follicular fluid composition. [1]

The average diameter of a recognizable refractile body under bright-field microscopy is approximately 10 µM. According to studies lipofuscin inclusions are associated with reduced fertilization and unfavorable blastocyst development only when their diameter is >5 µM. [1]

References:

  1. https://books.google.co.in/books?id=Kp5_AwAAQBAJ&pg=PA83&lpg=PA83&dq=refractile+bodies+in+oocyte&source=bl&ots=VCxnK7vkwu&sig=Akh6mi0lSgdMs36GtIulV5Zrgfo&hl=en&sa=X&ved=0ahUKEwi5mrDgspvcAhXIT30KHZkqAtI4ChDoAQhMMAc#v=onepage&q=refractile%20bodies%20in%20oocyte&f=false

ZP role in oocyte

The zona pellucid (ZP) is the specialized ECM layer that directly surrounds the oocyte. The ZP is an extracellular translucent matrix composed of long, cross-linked filaments, which first appears during oocyte growth and increases in thickness as oocytes increase in diameter. ZP represents the interface between the oocyte and its enclosing cumulus cells. ZP morphological abnormalities reported were centered in shape or thickness variety. ZP plays a critical role in fertilization by acting as a “docking site” for binding of spermatozoa followed by induction of the acrosome reaction in the zona bound sperm and an adequate block to polyspermy. Any disturbance in ZP morphology or texture may lead to abnormal fertilization result. Narrow PVS and heterogeneous ZP were always concurrences with the abnormal oocytes in many studies reported. [1] The ZP, acting as a protective and selective barrier, actually mediates the metabolic exchanges between the oocyte/embryo and the surrounding microenvironment [2]

A large number of ZP variants (appearance, thickness, irregularities, composition, and organization) have been described with the advent of ICSI. Thicker ZPs are associated with decreased fertilization rates, implantation and pregnancy rates. The ZP also plays a pivotal role in pre-implantation embryos; for instance, abnormalities in oocyte (and thus ZP) shape are associated with irregular cleavage patterns, compromised cell-cell contacts, and subsequent difficulties in the developmental progression. The importance of the ZP continues until the blastocyst stage, a time when the embryo needs to hatch out of the zona prior to implanting into the uterine epithelium. [2]

References:

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947078/
  2. https://books.google.co.in/books?id=zlB4T2ER4msC&pg=PA197&lpg=PA197&dq=PVS+in+oocyte&source=bl&ots=fw0PfSP80u&sig=e9QGojBuh79bRbFDr2fdrpBxCFI&hl=en&sa=X&ved=2ahUKEwjU9fyN7qzcAhXTXisKHaW1DHoQ6AEwCHoECAkQAQ#v=onepage&q=PVS%20in%20oocyte&f=false

PVS role in oocyte

The Perivitelline space (PVS) represents the acellular compartment in between the plasma membrane of the oocyte and its ZP. [2] The PVS of mammalian oocytes is made up of hyaluronan-rich extracellular matrix prior to fertilization. [1] It becomes clearly visible in a mature oocyte with the extruded polar body located in its most prominent portion. An indistinguishable PVS typically corresponds to immature oocytes while a distinct space to mature oocytes.

It has been proved that large PVS may result in disrupted or compromised communication between the cumulus cells and the oocyte, particularly via gap junctions and transzonal projections. Presence of large PVS will be seen due to over-mature eggs, where such eggs have shrunk in relation with ZP presenting a large gap between the oocyte and surrounding zona. [3] Large PVS is seen when the large portion of the cytoplasm is extruded together with the haploid chromosomal set during PB I formation. [2]   Oocytes with large PVS during ART treatment were usually reported with lower fertilization rate. [1] Granularity in the PVS has been associated with over-maturity of oocytes. Coarse granulation in the PVS is a morphological abnormality occasionally seen after stripping of the oocyte in preparation for ICSI and the presence of coarse granules in PVS is associated with lower pregnancy and implantation rates. [4 and 5]

References:

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947078/
  2. https://books.google.co.in/books?id=zlB4T2ER4msC&pg=PA197&lpg=PA197&dq=PVS+in+oocyte&source=bl&ots=fw0PfSP80u&sig=e9QGojBuh79bRbFDr2fdrpBxCFI&hl=en&sa=X&ved=2ahUKEwjU9fyN7qzcAhXTXisKHaW1DHoQ6AEwCHoECAkQAQ#v=onepage&q=PVS%20in%20oocyte&f=false
  3. https://www.researchgate.net/publication/229326639_The_oocyte?_sg=hXmowSf9g-nhMoxNcXfHz3khl6F7c6fBTyrlIkTAeAcVyFJR1kzxL4RsmxYM_31frJPpJ8sq8w
  4. https://link.springer.com/article/10.1023/A:1021243530358
  5. https://www.slideshare.net/Yasminmagdi/oocyte-morphology-assessment

Polar body role in oocyte

A polar body is a small haploid cell that is formed concomitantly as an egg cell during oogenesis, which generally does not have the ability to be fertilized. When certain diploid cells in animals undergo cytokinesis after meiosis to produce egg cells, they sometimes divide unevenly. Most of the cytoplasm is segregated into one daughter cell, which becomes the egg or ovum, while the smaller polar bodies only get a small amount of cytoplasm. [1]

Polar bodies eliminate one half of the diploid chromosome set produced by meiotic division in the egg, leaving behind a haploid cell. To produce the polar bodies, the cell divides asymmetrically, which later leads to furrowing (formation of a trench) near a point on the cell membrane. The presence of chromosomes induces the formation of an actomyosin cortical cap, a myosin II ring structure and a set of spindle fibers, the rotation of which promotes invagination at the edge of the cell membrane and splits the polar body away from the oocyte. [1]

Meiotic errors can lead to aneuploidy in the polar bodies, which, in most the cases, produces an aneuploid zygote. Errors can occur during either of the two meiotic divisions that produce each polar body but are more pronounced if they occur during the formation of the first polar body because the formation of the first polar body influences the chromosomal makeup of the second. For example, during the pre-division (the separation of chromatids before anaphase) in the first polar body can induce the formation of an aneuploid polar body. Therefore, the formation of the first polar body is an especially important factor in forming a healthy zygote. [1]

Oocytes showing an intact PB I give rise to higher rates of implantation and pregnancy, probably due to an increase in blastocyst formation. Recent research has shown that some polar body abnormalities may be an artifact of oocyte handling or aging. Abnormal morphology of PB1 in MII oocytes attributes to chromosomal aneuploidy of the oocyte. As PB 1 extrusion is directly related to spindle formation and sister chromatid exchange, it could be the most likely point of non-disjunction or aneuploidy formation. Fragmentation rates of the PB1 depend on the time elapsing between retrieval, denudation and ICSI performance. [2]

References:

  1. https://en.wikipedia.org/wiki/Polar_body
  2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663962/

Oocyte Granularity 

Granularity in ooplasm of the oocyte refers to the presence of “heterogeneous area”. Granularity is correlated with localization of mitochondria within the cytoplasm of the cell.

It also represents a domain of high ATP request which is very much necessary for normal development of embryos. A healthy MII oocyte should contain a clear and moderately granular cytoplasm. Top quality oocytes have the granularity homogeneously distributed at the sides of the oocyte cytoplasm and not at the center.

Oocyte with centrally located granular cytoplasm (CLGC) is considered as a dysmorphic oocyte. These kinds of oocyte show high chromosomal abnormalities like aneuploidy (presence of an abnormal number of chromosomes).

Also, the oocytes which lack granularity are considered as a bad oocyte. The severity of granulation is based on the diameter of the granular area, depth of lesion and crater-like appearance. [1,2 and 3]

References:

  1. https://www.researchgate.net/publication/229326639_The_oocyte?_sg=hXmowSf9g-nhMoxNcXfHz3khl6F7c6fBTyrlIkTAeAcVyFJR1kzxL4RsmxYM_31frJPpJ8sq8w
  2. http://atlas.eshre.eu/es/14611418225805670
  3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3455008/

 

Maturation Stage Of Oocyte

Oocyte nuclear maturity, as assessed by light microscopy, is
assumed to be at the MII stage when the PBI is visible in the PVS .

The MII stage is characterized by the alignment of the homologous chromosomes on the spindle equatorial plate during metaphase of the second meiotic division. It is generally recognized that 85% of the retrieved oocytes following ovarian hyper stimulation display the PBI and are classified as MII, whereas 10% present an intracytoplasmic nucleus called the ‘germinal vesicle’ characteristic of prophase I of the first meiotic division. Approximately 5% of the oocytes have neither a visible GV nor PBI and these oocytes are generally classified as MI oocytes. These oocytes may, however, be at the GV breakdown stage where the nuclear envelope has broken down but has not as yet progressed to true MI where the chromosomes are aligned on the metaphase plate in preparation for the completion of the first meiotic division.





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