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Exam Qs > And > Flashcards

And Flashcards

1
Q

Which gene in males inhibits Wnt4 expression in the undifferentiated gonad and promotes male sexual development?

A

Sry

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2
Q

When during gestation do cells migrate from the yolk sac to the gonadal ridge?

A

4-6 weeks gestation

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3
Q

In the female, which which duct regresses to allow for differentiation into the genital tract ( oviduct, uterus, cervix and upper one third of the vagina)?

A

Wolffian duct regresses ( mullerian duct remains and differentiates)

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4
Q

Organs in the female genital tract are composed of which 3 layers?

A

A protective outer layer, a middle wall of smooth muscle, and an inner mucosal layer

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5
Q

The pH of the cervix is higher, lower or same as the vagina?

A

higher (pH 7-8)

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6
Q

The cervix secretes buffers that..?

A

Neutralize the acidic environment of the vagina, increasing the pH from 4-5 to 6-7 at the opening of the cervix

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7
Q

MII oocytes have elevated levels of what substance that regulates the reduction of disulfide bonds between sperm chromatin-associated protamines allowing the sperm nucleus to decondense to support normal processing of the male pronucleus?

A

Glutathione

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8
Q

Plot used to see performance over time

A

Levey-Jennings Plot

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9
Q

expected-observed=difference
Differenced is summed together over tiem and should remain constant

A

Cusum plot (cumulative sum plot)

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10
Q

acceptable range for Sperm morph QC (levey Jennings)

A

+/-1 or 2 SD

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11
Q

CV%

A

SD/mean observed observations (usually 10-15% range)

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12
Q

PSA

A

Prostate-specific antigen
(enzyme secreted by the prostate gland to degrade structural proteins semenogelin I and II (secreted by seminal vesicles) during liquefaction 30-60 min post ejaculation

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13
Q

Which hormone aids in mucus production in the cervix to help sperm transport?

A

Estrogen

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14
Q

Uterine transport of sperm

A

uterine myometrium contractions

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15
Q

Sperm entry into the isthmus of the oviduct via the utero-tubal junction requires?

A

Correct sperm morphology, motility, and surface proteins (oviduct is free of immunological substrates and can maintain sperm viability for hrs or days)

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16
Q

Increased paternal age

A

reduced sperm motility and volume, increased DNA damage (DNA fragmentation); no observed reduction in concentration. Increased mutational load but no observed increase in aneuploidy

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17
Q

functional maturation involving process that does not alter the cell structure but changes its potential for fertilization

A

Capacitation. fert competence gained within the female reproductive tract

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18
Q

During capacitation, which changes in the plasma membrane prepare the sperm to undergo the acrosome reaction?

A

Shedding of proteins and cholesterol, post-translational and structural modification of proteins, change in tail movement (increase in the bend amplitude but a decrease in progressive movement)

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19
Q

Capacitation at the cellular level requires

A

the removal of inhibitory factors from sperm + alterations in membrane proteins and lipids

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20
Q

Capacitation at the molecular level

A

changes in intracellular pH, [Ca2+], and cAMP-dependent tyrosine phosphorylation/ dephosphorylation

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21
Q

sperm hyperactivated motility, ability to bind to the zone pellucida, and undergo the acrosome reaction are characteristics of what?

A

Capacitation.

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22
Q

Hyperactivation of sperm

A

occurs in the female tract and allows the sperm to move to the oocyte and penetrate viscous substances like mucus in the oviduct and through cumulous cells.

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23
Q

Capacitation in vitro

A

removal of sperm from seminal fluid for 2-4 hrs held in buffered media

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24
Q

long range guidance mechanism for sperm to find the oocyte

A

Thermotaxis- temperature gradient (lower in the isthmus and higher in the ampulla- gets warmer as closer to ovary and egg) guide capacitated sperm to fert site

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25
Q

short range guidance mechanism

A

Chemotactic method- may happen once sperm are closer to the egg (along with oviductal contractions) using chemoreceptors on the spermincluding olfactory receptors (chemoattractants may be secreted by follicle or mature egg)

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26
Q

Acrosome reaction is exocytotic meaning

A

things are released from sperm to the environment

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27
Q

induced following sperm binding to the ZP

A

acrosome reaction (must happen prior to sperm fusing with the oocyte (oolema essentially) happens at the ZP

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28
Q

Acrosomal contents are released when

A

the outer acrosomal membrane fuses with the the overlying sperm plasma membrane

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29
Q

Membrane bound organelle on sperm that originates from the golgi apparatus

A

acrosome

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30
Q

Hyaluranidase is present

A

sperm head- either plasma membrane or released from acrosome

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31
Q

What could zona binding tell you about the sperm or oocyte?

A

could tell you if the zona of the ova were abnormal or sperm that failed to bing were abnormal

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32
Q

guanine nucleotide-binding regulatory protein + PLCbeta1 and tyrosine kinase (TK) receptor coupled to PLCgamma

A

receptor-mediated signaling pathways in sperm plasma membrane involved in acrosome reaction caused by fusion with ZP

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33
Q

acrosome reaction by ZP caused by

A

progesterone and ZP proteins. Both lead to an increase in intra cellular Ca2+ within the sperm

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34
Q

What event occurs to induce depolarization of sperm membrane potential, increased intracellularpH, increased intracellular Ca2+ in the capacitated sperm?

A

ZP-sperm binding

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35
Q

ZP-sperm binding&raquo_space;fusion of sperm plasma membrane with outer acrosomal membrane»

A

release of acrosomal contents

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36
Q

ZP1, ZP2, ZP3, ZP4 can work as a complex

A

all 4 ZP proteins play a role in sperm egg binding and more than one ZP proteins induces the acrosome reaction

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37
Q

ZP1, 3, 4 bind to which part of the sperm?

A

capacitated head (pre-acrosome reaction)…that comes next

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38
Q

ZP2 binds to which part of the sperm?

A

the acrosome-reacted sperm

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39
Q

sperm penetrate the ZP through two mechanisms

A
  1. mechanical forces (motility)
  2. acrosomal proteases that hydrolyze the ZP glycoproteins
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40
Q

Role of ZP x4

A

spermbinding
induction of Acrosome rxn
acrosomal exocytosis
block polyspermy

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41
Q

2 blocks to polyspermy

A
  1. fast block: oocyte-membrane block&raquo_space;depolarization of the oocyte membrane following binding of first sperm
  2. Slow block: zona rxn/cortical rxn&raquo_space; hardening of ZP proteins by enzymes (hydrolyses, proteinases, peroxides) released in cortical granules after sperm-oocyte binding
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42
Q

can a sperm that has already started the AR still bind with the zona p?

A

yes- sperm binding is a redundant process-many ways for it to happen in case something goes wrong. other sperm surface proteins that can interact with the ZP

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43
Q

ROS can impact capacitation, hyper activation and AR induction? T/F?

A

true

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44
Q

Sperm in the PV space fuse where with the oolema?

A

fusion occurs btwn the post-acrosomal region of the sperm plasma membrane and the oocytes microvillous surfaces- except can’t bind where 2nd metephase spindle and 1st polar body are located

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45
Q

2 plasma membrane proteins essential for sperm-oolema binding

A
  1. Izumo-1(sperm) - must move from outer acrosomal membrane to mid plasma membrane region during AR (AR incomplete sperm cannot bind oocyte)
  2. Juno (oocyte)- shed from the oolema rapidly following fert and may be another mechanisms for polyspermy block
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46
Q

SPA

A

sperm penetration assay (zona-free hamster eggs can be fertilized by sperm from many mammalian species ) test for functionality of human sperm

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47
Q

Oocyte activation mechanisms induced by sperm

A

Sperm introduces a soluble cytosolic factor PLC(zeta), into oocyte cytoplasm acting as molecular trigger of activation causing cytoplasmic calcium oscillations

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48
Q

The cortical reaction

A

Receptor modification and Zona hardening and loss of sperm binding capacity

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49
Q

Protease Ovastacin and fetuin-B

A

cortical granule protease that hardens zona; inhibitor of ovastacin to sustain sperm binding

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50
Q

In spermiogenesis, somatic (non-gametic) histones in DNA are replaced with

A

Protamines, for chromatin condensation

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51
Q

what type of bond hold compacted DNA in place in elongated spermatids?

A

Disulfide bonds (formed by oxidation of the sulphhydral groups)

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52
Q

Syngamy

A

two adjacent pronuclear formation

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53
Q

First step in sperm nucleus decondensation?
what happens after sperm-oocyte fusion and demembrenated sperm are exposed to oocyte factors (in nuclear and cytoplasmicalaly mature oocytes)?

A

reduction of disulfide bonds btwn protamines by oocyte-derived disulfide bond reducer glutathione

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54
Q

Removed sperm protamines are replaced by what after sperm nucleus decondensation?

A

replaced by oocyte histones, organizing them into nucleosomes

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55
Q

reduction of sperm protamines

A

glutathione and heparin sulfate (protein acceptor)

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56
Q

sperm decondensation is dependent on which environmental factors?

A

time, pH, temperature

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57
Q

Sperm centriole becomes zygotic centrosome, needed for syngamy

A
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58
Q

SRY and SOX9 actions are required for testis determination

A

SRY on Y chromosome up regulates SOX9 (master testis determining factor)

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59
Q

Fetal leydig cells at 8 weeks (promoted by hCG) produce

A

testosterone, needed to for maintenance of wolffian ducts and masculinization of genitalia

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60
Q

Germ cells arrive at gonadal ridge at

A

5 weeks gestation

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61
Q

Exocrine function of testis

A

production of sprm, involving sertoli cells, serve a nurse cells and maintain the blood-testis barrier

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62
Q

endocrine function of testis

A

production and secretion of androgens from the Leydig cells (exocrine function is dependent on this endocrine function)

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63
Q

Paracrine function of testis

A

local controls of spermatogenesis through paracrine factors like cytokines, growth factors.

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64
Q

Leydig cells at 8 weeks gestation produce which hormone, neede for maintenance of wolffian ducts?

A

testosterone

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65
Q

From which embryonic tissue layer are wolffian ducts derived from?

A

Intermediate mesoderm - give rise to kidney and male genital tract (in the female, wolffian ducts regress at 2 months- same time as male leydig cells start producing T)

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66
Q

From which embryonic tissue layer are mullarian ducts derived from?

A

Coelomic epithelium - give rise to falopian tubes, ducts in pelvic region fure to form the uterovaginal canal

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67
Q

blood-testis barrier

A

Maintained by sertoli cells. tight junctions in sertoli cells serve to prevent proteins and antibodies from reaching spermatogenic cells and eliciting an immune response

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68
Q

FSH fxn in sertoli cell

A

Pituitary FSH stimulates receptors in sertoli cells to produce sertoli-derived inhibin that makes negative feedback loop to inhibit GnRH>FSH and activin release

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69
Q

Testosterone fxn in sertoli cell

A

androgens are ABSOLUTELY required for spermatogenesis. androgen receptors on sertoli cells facilitate spermatogenesis (no androgen receptors on germ cells- all actions of anderogens are on the sertoli cells for sperm production)

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70
Q

Spermiation

A

release of mature sperm into the lumen of the seminiferous tubule

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71
Q

T/F - # of sertoli cells dictates the spermatogenic potential of a man?

A

True. > sertoli cells = > # of sperm that can be produced

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72
Q

Blood-testis barrier

A

Tight junctions (not gap junctions) between neighboring sertoli cells at the basal laminae of the lumen.

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73
Q

Basal compartment of seminiferous tubule

A

between sertoli junctions and basal laminae containing the early spermatogonia

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74
Q

adluminal compartment of seminiferous tubule

A

above the sertoli cell tight junctions containing more advanced germ cells

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75
Q

Sertoli cells produce which form of inhibin, to provide negative feedback for FSH production?

A

Inhibin B

(if damage to germinal epithelium (sertoli cells), results in increased FSH seretion.

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76
Q

Sertoli cells produce which hormone during fetal development to regress the mullerian ducts?

A

AMH

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77
Q

Leydig cells primary function

A

steroid hormone makers- synthesize steroid hormones from cholestrol

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78
Q

Leydig cell androgen production is stimulated by

A

pituitary LH

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79
Q

rate limiting step of steroidogenesis in leydig cells

A

mobilization of cholesterol from intracellular stores to leydig mitochondrial membrance where it is converted into pregnenolone

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80
Q

Which protein is responsible for transporting cholesterol into mitochondria?

A

acute steroidogenic regulatory protein (StAR)

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81
Q

testosterone is reduced in peripheral tissue to dihydrotestosterone (DHT) by which enzyme?

A

5alpha reductase

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82
Q

testis also produce estrodiol from conversion of testosterone to estrogen by which hormone

A

aromatase (CYP19)

(estrogen receptor found in leydig cells-estrogens may regulate testosterone production)

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83
Q

Where are Leydig cells located?

A

between seminiferous tubules inthe interstitial space close to blood vessels and lymphatics

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84
Q

When are Leydig steroidogenically cells active?

A

8-18 weeks fetal develpoment and puberty onward

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85
Q

Leydig cell function is regulated by which two hormones

A

LH: stimulates T biosynthesis
Prolactin: induces expression of the LH receptor

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86
Q

Which germ cell population are undergoing meiosis?

A

spermatocytes

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87
Q

spermatogonia are

A

slowly dividing stem cell population

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88
Q

which germ cell population are already haploid and undergoing morphological changes?

A

spermatids

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89
Q

order of spermato -

A

goina»cytes»tids

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90
Q

Which cells in the body have the highest rate of mitosis?

A

Spermatogonia in the testes

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91
Q

spermatogonial stem cells

A

in basal compartment of blood-testes barrier next to sertoli cells and can can regenerate post toxic insult

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92
Q

Type A spermatogonia

A

least mature sperm cells located directly along basal lamina. require retinoic acid to convert type A to type B. Type A spermatogonia are connected my small cytoplasmic bridges

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93
Q

2 types of Type A spermatogonia

A

A pale: spermatogonia
A dark: considered to be stem cells

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94
Q

Type B spermatogonia are more differentiated than Type A?

A

True

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95
Q

What is needed to begin meiosis in sperm?

A

retinoic acid acts directly on spermatogonia to stimulate entry into meiosis which creates Type B from Type A

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96
Q

What is the ploidy of Spermatogonia

A

Diploid. Located along basal lamina of seminiferous epithelium. (outside of blood testis barrier- ie wont cause issue because still diploid cells, on the blood side of the tight junctions between sertoli cells)

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97
Q

Meiosis in sperm cells:

A

Type B spermatogonia> enter meiotic pathway

DNA replication> 4N (tetraploid gamete)

introduction of genetic variation of gametes from recombination, double-stranded breaks and crossing over, DNA repair

> 2 successive reductive devisions» haploid spermatids

4N/ 2N/ 2= 1N (haploid -23 chromosome spermatids)

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98
Q

Primary spermatocytes (4C) characterized by

A

large, 4C chromosome value, now have moved into the adluminal compartment inside the blood-testis barrier as non-diploid cells. Will then divide into 2 secondary spermatocytes (2C DNA content)

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99
Q

Secondary spermatocytes(2N)

A

2C; formed by completion of first meiotic division, will undergo one more division to form 2 sister spermatids with haploid (1N- 23 chromosomes) chromosome content

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100
Q

Which stage do germs cells end in at the completion of spermatogenesis (genetic component- end of meiotic divisions)

A

Spermatids

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101
Q

Spermiogensis is the process of

A

maturing of sperm cells
spermatids» mature spermatozoon

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102
Q

3 phases of spermiogenesis:

A
  1. Development of the acrosome: development of acrosomal cap where storage of hydrolytic enzymes (golgi stage, cap phase, acrosomal phase, maturation phase)
  2. Maturation phase: nucleus elongates, cytoplasm is phagocytosed by sertoli cell, spermatozia released into lumen
  3. Condensation of the nucleus: somatic chromatin histones are replaced by highly basic protamines, chromatin fibers condense and compact nulcear material into a tightly packed structure, transcriptional activity stops (packed up into the suitcase until get to final destination)
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103
Q

What part of the sperm contains hydrolytic enzymes?

A

The acrosome- the acrosome contains hyaluronidase, neuraminidase, acid phosphatase, and trypsin like protease needed by the mature sperm to transverse the cumulus complex and zona pellucida

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104
Q

sperm flagellum is formed by what
?

A

one of the sperm centrioles differentiates to form flagellum (mid piece of the tail is thickened region of flagellum where movement is generated. mitochondria migrate along developing axoneme)

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105
Q

What is the flagellum made of?

A

axoneme (9+2 microtubule doublets in a concentric arrangement) surrounded by fibrous sheath and keratin containing dense outer fibers. Mitochondria in mid-piece provide energy

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106
Q

Sperm Type A can be pale (Ap) or dark (Ad). Which type goes on tp make Type B?

A

Pale. Type Ap go on to make 2 Type B through mitotic division, which will then progress to enter meiosis.

Type Ad, dark, can either produce type Ap(pale) or more Type Ad (dark-stem cell)

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107
Q

Spermiogenesis

A

differentiation of the round spermatid to undergo morphological changes to develop an acrosome and flagella and undergo nuclear condensation and extrusion of cytoplasm

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108
Q

tetratozoospermia

A

low % normal morphology

Example:

round headed sperm/ globospermia due to the absence of the acrosome or acrosome atrophy

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109
Q

Length of the cycle of spermatogenesis (spermatogonia to spermatozoa)?

A

64 days

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110
Q

on diagram be able to define

A

radial spoke, central sheath, dynein arms, subfiber A and B

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111
Q

Factors that can decrease motility

A

tobacco, sexual lubricants, saliva, alcohol and drugs

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112
Q

What powers the dynein arms in sperm axoneme to move

A

ATP hydrolysis

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113
Q

Which stages of spermatogenesis are testosterone dependent?

A

Transition from primordial germ cell to type A spermatogonia during fetal development (meiosis), reductive devision of primary to secondary spermatocytes (4N»2N»1N) (meiosis), spermiation (sperm maturation)

(non-mitotic division)

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114
Q

What is the role of Thyroxine in fetal gonad development?

A

hormone/factor that regulates Sertoli cell numbers during development

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115
Q

Abnormalities in the pampiniform plexus, network of interconnected veins to cool arterial blood entering the testis, such as varicocele, causes what?

A

adverse effects on spermatogenesis by interfering with cooling mechanisms of the plexus

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116
Q

what is a varicocele

A

vericose veins in the spermatic cord of the scrotum

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117
Q

Where in the male tract do sperm acquire motility?

A

Epididymis (head-caput, body-corpus, cauda-tail).

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118
Q

How long does it take for sperm epidydimal maturation(to the head of the epididymis)?

A

10 days

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119
Q

How long until sperm reach the end of the epididymis and are ready for ejaculation?

A

16 days (so 90 days from start of spermatogenesis until ready for ejaculation) so give 3 months from insult to see if sperm quality reappears)

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120
Q

sperm go from

A

seminiferous tubule»
lumen»
caput (head) epididymis»
corpus(body) epididymis»
cauda(tail) epididymis»
vas deferens»urethra

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121
Q

While maturing in the epididymis sperm change by

A

gain motility, acquire additional surface glycoproteins, change net surface charge, increased capacity for glycolysis, increased ability to adhere to ZP, changes in immunoreactivity) these changes are regulated by androgens

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122
Q

release of sperm from cauda epididymis by

A

short adrenergic contractions by cauda epi. and vas def during ejactulation

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123
Q

emission vs ejaculation

A

The ejaculation process consists of two phases: emission (when semen is created by fluids from the prostate, seminal vesicles, and vas deferens and released into the penis) and expulsion (which follows the emission phase as the ejaculation climaxes).

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124
Q

Summary of epidydimal fxn

A
  1. sperm maturation
  2. stabilization of condensed chromatin
  3. changes in surface charge of the plasma membrane
  4. new sperm surface proteins
  5. sperm storage
  6. sperm transport by peristalsis
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125
Q

spermatic cords contains

A

vas deferens, cremaster muscle, spermatic artery and veins (pampiniform plexus)

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126
Q

Prostate

A

largest accessory gland (contributes 15-30% or 0.5mL to semen)clear, slightly acidic fluid (high citric acid content to maintain osmotic equilibrium in semen). Prostatic fluid contains enzymes required for liquefaction of the ejaculate coagulum.

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127
Q

Seminal vesicles

A

Fluid from seminal vesicles contributes 45-80% (2-2.5mL) of the volume of the semen. Rich in fructose (major source of glycolytic energy for the sperm) and prostaglandins. contain enzymes required for formation of coagulum, androgen dependent organ.

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128
Q

What can elevated or absent levels of fructose in semen mean?

A

Function of seminal vesicles or ejaculatory duct disfunction/obstruction in azoospermic men. no fructose may mean congenital bilateral absence of vas deferens and seminal vesicles

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129
Q

T/F vas deferens and seminal vesicles are derived from sam embryonic origin

A

true. if vas deferens is congenitally absent, seminal vesicles and fructose in semen will also be absent

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130
Q

seminal prostaglandins do what?

A

urethral muscle contractions during ejaculation and may aid sperm transport through female repro tract.

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131
Q

Cowper (bulbourethral) gland and Littre glands secrete?

A

first fluid portion of ejaculate. 0.1-0.2mL, low volume to coat/lubricate urethra, clear fluid rich in mucoproteins

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132
Q

Seminal plasma

A

combination of fluids from all accesory sex glands

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133
Q

semen

A

seminal plasma + spermatozoa

134
Q

Order of seminal vessicle emission

A

Cowpers&Littre»prostate»ampulla»epididymis, and finally the seminal vesicles

135
Q

Which portion of the ejaculate contains the sperm?

A

first portion (so loss of this portion will result in inaccurate counts)

136
Q

Coagulum takes how long to liquify?

A

Liquefaction usually in 5-20 min (up to 1hr)

137
Q

No coagulum?

A

Seminal vesicle issue

138
Q

No liquifaction/

A

Prostate issue

139
Q

Varicocele

A

varicose veins in pam. plexus, can affect spermatogenesis due to temperature regulation. usually found on the left side. can be palpated in 40% of men with oliogospermia (but also present in normal men ?)

140
Q

Kartagener Syndrome

A

No sperm motility due to asthenospermia. Genetic disorder affecting cilia. No dynein arms in the axoneme so no sperm movement in tail) also will have a history of sinus and pulmonary issues. (Do a live/dead stain to prove dead not just immotile)

141
Q

Cryptorchidism

A

failure of the testis to descend into the scrotum during fetal development. (uni or bilateral) 6% of births. if not decended by 6 mo, surgical intervention required.

142
Q

Retrograde ejaculation:

A

incomplete bladder neck closure caused by dysfunctional internal sphincter during ejaculation. Semen is ejaculated into bladder instead of out urethra. severe hypospermia. Patients must collect post ejaculation urine sample that is washed with a buffer otoisolate sperm.

143
Q

Hypogonadotropic hygonadism (Kallmann Syndrome)

A

Low GnRH» decreased serum Testosterone, LH, and FSH. Decreased sense of smell Oligospermia or azoospermia (little or now sperm) can be treated with hCG to stimulate androgen production and gonadotropins to provide FSH for spermatogenesis. Possible with these treatments to forgo ART and achieve pregnancies.

144
Q

Spermatogenic Failure (Cryoptozoospermia, Hypospermatogenesis, early or late maturation or meiotic arrest) seroli-cell only syndrome

A

Sertoli cells are present but germinal epithelium is absent, sperm are rarely present. sertoli cell dysfunction: no inhibin B production, so no negative feedback on FSH, so elevated serum FSH levels. No effect on leydig cells, so normal LH levels

145
Q

Recent WHO manual editions recommend semen samples should be collected …

A

2-7days post abstinence

“48h but not longer than 7 days” post previous ejaculation

146
Q

New sperm specimen containers should be tested before use by?

A

Checking motility hourly for 4 hr against control

147
Q

WHO 5th addition says semen volume should be measured by?

A

Weight not volume (use pre-weigh collection cup- g to mL)

148
Q

What is an acceptable form of ID for dropoing off a semen specimen?

A

divers license

149
Q

Liquefaction

A

Not the same as viscosity. Should occur within 0.5-1h. (non-liquefaction may indicate prostatic dysfunction)

150
Q

Viscosity

A

measured by drawing up and releasing semen (stringy-ness)
1-4 scale (normal> 1 slight stringy> 2 mod. stringy> 3 difficult to draw up> 4 cant be drawn into pipette (semen solid mass)

151
Q

Hyperviscosity can be broken down

A

passing thorugh a wide bore needle, chemically using 0.2% alpha-amylase or chymotrypsin

152
Q

Semen volume

A

measured by weight under the assumption that the density of semen is 1g/mL

153
Q

Normal semen volumes

A

WHO 4th: >2.0 mL
WHO 5th: >1.5 mL

upper limit 5.5 mL

154
Q

Hypospermia

A

low semen volume <1.5mL (ejaculatory dysfunction, collection error, reduced abstinece period)

155
Q

Hyperspermia

A

increased semen volume (>5.5mL) may be indicative of increased abstinence

156
Q

Aspermia

A

(no semen) volume 0.0mL
may indicate retrograde ejaculation

157
Q

Color & turbidity

A

translucent or whitish gray in color (yellow maybe urine in sample)

158
Q

hematospermia

A

blood in semen (pinkish)

159
Q

pH of semen

A

7.2-7.8 (8.0) (litmus paper)

160
Q

pH of semen can be affected by

A

seminal vesicle secretions

161
Q

Wet mounts are used for

A

subjective analysis of sperm counts, motility, morphology, kinetics, epthelial cells, rounds cells (WBC or immature sperm) and agglutination

162
Q

Agglutination

A

sperm adhering to other sperm, mucus strains, or non sperm debris

Reported on a scale 1>4
absent >1occasional > 25% or less >3 25-50%> 4+ gross agglutination (few free swimming sperm)

163
Q

WHO 5th addition grades of agglutination

A

degree 1-4 and site of attachment (grades A-E)
1. isolated (<10 soerm per agglutinate)
2. moderate(10-50 sperm per agglut.)
3. large (>50 sperm per agglut.)
4. gross (all sperm agglutinated and agglutinates connected)

A. head-head
B. tail-tail
C. tail-tip-to-tail-tip
D. mixed
E. tangle (all enmeshed)

164
Q

Agglutination may be indicative of presence of?

A

Anti-sperm antibodies

But not always, non-specific agglutination is common

165
Q

Subjective vs objective methods to asses sperm motility

A

subjective- straight wet mount
Objective- specially designed counting chambers like makler, disposable counting chambers or CASA

166
Q

Sperm motility

A

WHO 5th : total motility (PR+NP)=%40 and lower ref limit for PM =32%
Previous WHO editions consider >50% motile normal

167
Q

Asthenozoospermia

A

decreased sperm motility <50%)

168
Q

Sperm vitality

A

can be determined by eosin vital staining red=dead. Can be used to determined truly dead sperm (necrozoospermia) from non-motile.

Should be performed if motility is less than 30-40% to tell difference btwn dead and immotile

169
Q

Sperm kinetics

A

ability for sperm to move in progressive forward manner, velocity
subjective (none, poor, fair, good, exc) (0 none»4 exc)

170
Q

CASA computer assisted semen analysis

A

increased objectivity and consistency of measurements but can also miscalculate (ideal for specimens 20-50X 10^6 mL

171
Q

hemocytometer for sperm count

A

oldest and most used method for counting sperm
100um deep recommended by WHO 5th

172
Q

Avg normal sperm count minimum?

A

> 20x 10^6/ mL but lowering to maybe >15

173
Q

Oligozoospermia

A

low count
<20x 10^6/ mL, or <15x 10^6/ mL (WHO 5th)

174
Q

Polyzoospermia

A

high count
>250x 10^6/ mL

175
Q

Azoospermia

A

no sperm found in ejaculate

Confirmation of azoospermia should be determined by post-centrifugation @ 1000g for 15 min and a wet prep

Check for seminal fructose (CBAVD)
check FSH levels to rule out testicular failure (elevated FSH at 3x normal levels in testicular failure patients)

176
Q

Cryptozoospermia

A

so few sperm only found after centrifugation (not the same as true azoospermia)

177
Q

Sperm morphology

A

must be assessed using stain
WHO 3rd: >30%
WHO 4th: >14%
WHO 5th: >4%

178
Q

-spermia

A

refers to semen

179
Q

-zoospermia

A

refers to sperm

180
Q

Are immature sperm counted in sperm counts?

A

no

181
Q

Globospermia

A

round headed spermatozoa lacking an acrosome

182
Q

Teratozoospermia

A

abnormal sperm morphology

<30% WHO3rd
<14% WHO4th
<4% WHO5th

183
Q

Calculating concentration of round cells in a wet prep relative to the number of actual sperm

A

C= N x S/100

C= concentration of Round cells
N= number of other cells counted in the same field as 100 sperm
S= sperm concentration in x10^6/mL

ex. 10 WBC per 100 sperm with sperm count of 120x10^6

10 x 120/100 = 12x10^6 WBC/mL

184
Q

Pyospermia

A

> 1x10^6 WBC/mL, may be indicative of infection

185
Q

PVSA

A

post vasectomy semen analysis

-8-16 weeks post vasectomy
-fresh specimen with <100,000 non-motile sperm
-vasectomy considered unsuccessful if motile sperm found 6 mo. post procedure

186
Q

Between the 2nd -4th edition of the WHO manual, which semen parameter changed?

A

motility

187
Q

Best method to test sperm count reproducibility? (sperm count QC)

A

accubeads

188
Q

Levey-Jennings plots

A

plot accubead counts for sperm count QC

y -axis: count
x-axis: assay number
Can see over time if counts fall out of upper or lower limits (over counting or undercounting trends)

189
Q

Cumulative sum (cusum plots) for sperm count QC

A

y-axis: + or - values from 0 (differences from expected)
x-axis: day #

accubeads counted on a daily basis.
expected value - actual observed value
this difference is summed together over time
the cumulative sum over time is plotted to show trend. Linear trend if assay is in control, slope up or down if out of control

190
Q

Sperm morphology QC

A

Levey-jennings plot (can plot when things fall within or outside pre-established upper and lower ranges)

Acceptable ranges can be set as +/- 1 or 2 SD of the mean
(+/- 2SD =95% of population lies)

191
Q

Sperm morphology QC using Intra-laboratorian CV%

A

% coefficient of variation (%CV)= SD/mean of observations then plot the %CV over time

(the acceptable %CV is set by the lab, usually 10-15%)

192
Q

Sperm motility QC

A

use pre-recorded videos of moving sperm (video better than wet mounts bc greater repeatability to be sure people are seeing the same thing)

193
Q

Role of sperm function tests

A

detect abnormalities of fertilization potential not detectible in semen analysis

194
Q

Hypo-osmotic swelling test (HOS)

A

Can determine if immotile sperm are alive with an intact membrane with fluid transport capabilities. simple viability/functionality test.
normal if tail swelling is >60%
Tail should swell in hypo-osmotic conditions (does the membrane work correctly to compensate for osmotic change?)

195
Q

Sperm-cervical mucus contact (SCMC)

A

Place semen and preovulatory mucous on a slide next to each other mix together and observe under microscope. If sperm are shaking then it is considered a “positive” for anti-sperm antibodies. Determine % shaking:

negative (0-25% shaking)
weakly pos. (26-50%)
Positive (51-75%)
Strongly pos. (76-100%)
(crossover test can be done with donor semen or donor mucous to see if male or female partner issue)

196
Q

DNA fragmentation tests (not currently routine recommendation by ASRM)

A

how well DNA content of sperm is maintained (intact)
-Acridine orange staining (AO)
-sperm chromatin structural assay (SCSA)
-TUNEL
-Halosperm (stain with halo around sperm head if normal and not DNA frag)
-Comet Assay
All these tests measure DNA integrity accept AO stain

Control for this assay is to induce ROS damage by putting control sample with hydrogen peroxide (>27% frag considered abnormal but no standardization)

197
Q

DNA frag in sperm may be caused by

A

deficiency in DNA/chromatin packaging, ROS in tract, shear forces of ejaculation, deficiency of spermiogenesis, exposure to toxins

198
Q

What is the endpoint to know that capacitation has taken place?

A

The acrosome reaction

199
Q

How can the acrosome reaction be induced in vitro?

A

By exposing the sperm to Calcium ionophores (Acrosome reaction to Ionophore challenge) ARIC or to progesterone. ARIC is also used to identify globozoospermia (no acrosome present, round head)

200
Q

How can you measure if the acrosome reaction has taken place?

A

It can be measured using fluorescent dyes and microscopy. If acrosome is intact- AR has not taken place. If banded or absent, AR has occured. (be careful if all blank-maybe globozoospermia with no acrosome at all) May provide information about fertilization potential.

201
Q

How is the ARIC scored?

A

calculate the % of sperm AR in test sample vs control. Normal is >15%, abnormal is <10%

202
Q

What can you learn from a sperm binding assay?

A

Either that the zona is abnormal or that the sperm is abnormal in binding capacity.

203
Q

Sperm zona binding assay

A

Zona binding capacity of Patient and donor sperm are compared using a bisected zona from the same source.

If patient sperm dont bind» sperm issue
If they can to any zona» patient zona issue

if >35% ration of patient/donor binding considered normal

204
Q

Sperm penetration assay (SPA) (Zona free hamster egg penetration assay )

A

Test measures ability of sperm to undergo capacitation, fuse with membrane, and decondense chromatin to form a male pronucleus

After removal of the zona, hamster eggs can be penetrated (fertilized) by sperm of many species.

+ score indicates high likelihood of success with IVF
- score cannot predict failures resulting from Zona binding issues or egg factors….

205
Q

Sperm FISH

A

fluorescence in situ hybridization for chromosome aneuploidy

Fluorescently labeled probes specific to centromeres of chromosomes 13, 18, 21, X and Y were hybridized to decondensed sperm heads.
Different colors assigned to each chromosome to distinguish haploid and diploid cells and presence of mono- or disomies of different chromosomes

Can be done for couples with reoccurring pregnancy loss, or severely oligospermic, or previous history of offspring with monosomy or trisomy syndrome

206
Q

Non-disjunction

A

meiotic abnormalities resulting in chromosomal aneuploidy (abnormal numbers of whole chromosomes)

either disomic (2 chromosomes) or nullisomic (0 chromosomes) for a particular chromosome.

if an embryo were created with these sperm they would be trisomic (3 copies) or monosome (one copy) leading to miscarriage or birth defects

207
Q

What are some examples of diseases that can be caused by non-disjunction (gain/loss of whole chromosome)?

A

Turner (X), Down’s (tri 21), Patu, Edwards, Klinefelter’s syndromes

All monosomies or trisomies

208
Q

Antisperm antibody (ASAB)

A

immunologic infertility. Antibodies can be found in men and women. Confusing etiology- can be in tract or in blood serum. Caused my immunologic response to sperm or other proteins etc that come in contact with immunologically active cells, creating an immune reaction to sperm.

in men: can be caused by physical trauma to blood-testis barrier (ex most commonly vasectomy or cystic fibrosis)

in women: maybe caused by disruption of mucosal layer of genital tract allowing antigens to come into contact with seminal products

209
Q

Pathogenesis of ASAB

A

When ASAB are present, the bind to the surface of the sperm cells and impair motility and function (immobilizing type ASAB). May inhibit capacitation.

Can cause clumping of sperm — agglutination (agglutinizing type ASAB)

ART procedures have mostly eliminated this issue

210
Q

Diagnostic tests for ASAB detection

A

Usually some type of visual determination of degree of agglutination or immobility

Serum added to sperm and but on either gelatin, slide, IgA, IgM, IgG beads, and agglutination or motility is observed verses control.

211
Q

How long after aspiration should ICSI or IVF be performed?

A

generally 4-6 h, however some success with 2 h.

(ovualtion occurs approx 36 h post-hCG with most oocytes having reached MII)

212
Q

How long should sperm be co-incubated with oocytes for IVF?

A

overnight with evaluation for fertilization between 14-18 h post insemination.

213
Q

What is the average concentration of sperm added in conventional IVF?

A

50,000 -150,000 sperm per oocyte/mL

(Can increase if poor semen parameters- the art in ART)

other references say 200-250K/mL

214
Q

sperm concentration for IVF calculation?

A

sperm conc. desired/sperm conc. of spec (x 10^6)/volume in mL

(how does # of oocytes factor into this?) It doesn’t really…

fert rate should be >70% with polyspermy rate <5%

215
Q

What are the two components of semen?

A

Cellular fraction and liquid fraction

216
Q

How long can sperm be exposed to semen before exhibiting diminished fertilizing capacity?

A

30 minutes

sperm will die rapidly after 1 -2 h exposure to seminal plasma

sperm should isolated from seminal plasma within 1 hr of ejaculation and placed in nutritive buffered medium

217
Q

Recovery and concentration of motile sperm accomplished what?

A

should be done in a way to minimize sperm damage

removes seminal components including decapacitation factors, hormones and pathogens, ROS, anti-sperm antibodies,

218
Q

Methods for sperm separation from semen

A

density gradients,
pelleting (simple wash centrifugation)
swim up, swim down, swim out
filtration columns (glass wool)
transmembrane migration (Zymot)

Not all patient specimens react the same to all techniques (differences in recovery efficiency)

219
Q

Cons of centrifugation?

A

may damage sperm and consentration ROS

220
Q

What physical characteristics of semen could interfere with recovery efficiency?

A

viscosity, incomplete liquefaction, excessive numbers of non-sperm cells

221
Q

3 most common methods of sperm isolation from a fresh sample

A

Density gradient separation- increasing gradient layers of a colloidal suspension of silica particles. Less dense and nonmotile sperm caught between the layers

Swim up methods- rely on motile sperm to swim into a overlaying layer of culture media (CO2 or buffered media for 1 hr)

Sperm pelleting- simple wash (concentrate all cellular portion of semen (motile and nonmotile) Can be used for oligo- patients

222
Q

T/F
Increased centrifugation speed/time can lead to sperm damage?

A

True

223
Q

Is there one “best way” to separate sperm from semen?

A

No. Patient/circumstance dependent
(all prep techniques have been found to increase the DNA frag rate compared to raw semen samples)

Swim up samples after pelleting or gradient (ie motile sperm out of a pre-selected group) had lowest DNA frag levels — idea behind zymot?

224
Q

what is the normal centrifuge setting for gradient separation techniques?

A

20 min at 300x g

225
Q

Most viral reduction steps in sperm prep (ie for HIV patients) include which step?

A

Swim up, to separate sperm from leukocytes in pellet. Need to be post processing titers to be analyzed by PCR prior to use in ART

use in sero-discordant couples dictated by US State law

226
Q

Cryopreservation of sperm capacitates sperm faster than in a neat specimen so ready for use immediately. T/F?

A

true. Causes changes in sperm cell membranes causing a less heterogenous capacitation waves- all will be capacitated sooner SO can be used for IUI or IVF sooner

227
Q

Commercial sperm banks screen donors for?

A

disease/genetic status
routine semen parameters

228
Q

retrograde ejaculates look the same as normal?

A

No, often reduced motility due to exposure to acidic environment

229
Q

IUI preps

A

0.5-1.0 mL volume
(most important pre-wash parameters in order:
PM%
COnc
Total #
morph

In post-wash specimens, most important is TOTal # progressive
min # <1-2 million (our current is 5M)

230
Q

In vivo, how do sperm capacitate?

A

during passage through the female tract (removal of seminal decapacitation factors)

231
Q

Events associated with capacitation

A

removal of surface proteins

efflux of unesterified cholesterol from the sperm cell membrane

alteration of sperm proteins (tyrosine phosphorylation)

232
Q

WHat can affect the time course of capacitation?

A

temperature, energy metabolism and pH

233
Q

Common starting sperm concentration for traditional IVF is?

A

50,000 motile sperm per mL per Oocyte but can often be less

If desired target # is 50,000 sperm in 1000uL (1.0 mL), but volume is only 100uL drop, how many do you add? (see what the ratio of 50,000/1000uL = 50

so x/100=50 … 50*100=x = 5,000 sperm

234
Q

Why can’t ICSI guaranteen successful fertilization?

A

The integrity and status of the oocyte will ultimately determine whether it can be fertilized and activated (incomplete nuclear maturation? even if meiotically mature)

235
Q

POlyspermia

A

possible improper cortical granule populations and sperm blocking capabilities

236
Q

MSOME and IMSI

A

Motile sperm organelle morphology examination

Differential contrast microscopy in real time at or above 6300X allowing for selection for visible morphological defects

to then be used with intracytoplasmic morphologically selected sperm injection (IMSI)

237
Q

Capacitation and acrosome reaction completion are NOT required for successful fert by ICSI

A

True. the acrosome is shed within the ooplasm

238
Q

What % of men evaluated for infertility are azoospermic?

A

5-10%

obstructive azoospermic
non-obstructive azoospermic
retrograde ejaculation

239
Q

Obstructive azoospermia (OA)

A

physical blockage in the repro tract

Normal testicular sperm production but cannot be released

abnormality in the epididymis, vas deferens, or ejaculatory ducts (previous abdominal surgery, pelvic infection, failed vasectomy reversal, CAVD)

High likelihood of sperm after surgical retrieval

240
Q

Non-obstructive azoospermia (NOA)

A

severely impaired or non-existent sperm production. (usually diagnosable from endocrine dysfunction)
-primary testicular failure
surgical sperm recovery low as <50%

Causes:
-LOw T
-Low FSH
-genetic anomalies (gene deletions on Y chromosome governing germ line dev.)
-physical destruction of spermatogenesis
-severe gonadotoxic event (pre-pubertal viral infection, environmental toxins)

241
Q

TESA

A

Testicular Sperm Aspiration (A at the end = aspiration)
Tissue is removed by suction using wide-bore needle and contents flushed into search dish (office procedure under local anestesia)
presence of motile and immotile sperm

242
Q

TESE

A

Testicular Sperm Extraction
Seminiferous tubule is excised and biopsied tissue is dissected to check for sperm

(most invasive of the surgical sperm retrievals)

sperm recovered from testicular tissue may display no motility, occasional tail movement twitching or vibration
The sperm are immature and incapable of undergoing capacitation so ICSI required

243
Q

MESA

A

Surgically exposed tubule of the epididymis and sperm are aspirated using a needle

Should be used with ICSI due to reduced sperm parameters

For OA patients, sperm can be aspirated directly from the epididymis

Be sure to remove all red blood cells, sometimes using hypotonic lysis buffer to help remove blood cells, centrifuge and remove supernatant, sperm should be in pellet

244
Q

PESA

A

percutaneously collected with fine needle aspiration through the scrotum

245
Q

Exogenous sperm motility enhancers

A

-Pentoxifylline (inhibits phospodiesterase&raquo_space; raises intracellular cAMP levels ) use 3-5mM
-Caffine
-2-deoxyadenosine
-follicular fluid

For all of these, motility enhancement is transient

246
Q

What happens in the oocyte after fertilization?

A
  1. Intracellular Ca 2+ release
  2. meiotic reactivation
  3. separation of chromatids
247
Q

Cellular events that culminate in pronuclear development

A

-sperm penetration of oolema
-oocyte activation
-sperm head detachment
- sperm cell DNA condensation and remodeling

248
Q

What happens when the sperm penetrates the oolemma to induce oocyte activation?

A

Internal Ca2+ mobilization, alteration of oolemma electrical potential, completion of metaphase II, signal transduction, exocytosis

249
Q

What happens after sperm head detachment in the oocyte?

A

-decondensation of sperm cell DNA and packing proteins
- sperm centriole deposition
-oocyte chromosome mobilization and decondensation
-pronuclear membrane merging
-microtubule formation to drive pronuclear migration

250
Q

Syngamy

A

pronuclei found in close, direct opposition followed by syngamy ‘fusion of gametes’ however not truly fusion:
dissolution of closely opposed pronuclear membranes and progression of both maternal and paternal chromosomes into DNA replication for cell division.

251
Q

Markers of correct fertilization

A

2 pronuclear structures structures contains several nucleoli and presence of 2 or more polar bodies.

(use DIC with glass dishes or hoffman optics with plastic dishes)

male PN slightly larger than female PN

252
Q

Which morphological aspects of fertilization markers can be used to determine quality?

A

-pronuclear morphology (distinguish from vacuoles)
-polar body orientation can predict preimplantation embryonic development and implantation potential
-cytoplasmic halo - should see
- debris in PV space
- refractile bodies in cytoplasm

253
Q

A normally fertilized oocyte will have

A

2 pronuclei and 2 polar bodies

abnormal (1PN, 1 PB = PB2 retention or parthenogenic activation)

abnormal (1PN, 2 PB = sperm decondensation does not occur)

abnormal (3+ pronuclei, 2PB) polyspermy

254
Q

Fert failure after conventional IVF

A
  1. sperm-associate failure to bind/penetrate zona (AR failure, capacitation, receptor-binding failure, Antisperm antibody)
  2. Sperm did penetrate zona and oolemma BUT incomplete DNA condensation (incomplete oocyte cytoplasmic maturation or sperm-associated decondensation failure)
  3. Zona abnormalities
255
Q

Fert failure after ICSI

A
  1. technique failure on the part of the technician
  2. oocyte dysfunction
    3.Sperm tail membrane is insufficiently damaged (damage improves oocyte activation and sperm deconensation
  3. improper PB/needle positioning at injection (can disrupt the 2nd meiotic spindle during needle entry)
256
Q

Oocyte dysfunction

A

incomplete cytoplasmic maturation, oocyte activation failure (oocyte or sperm related), inability for sperm to condense to be processed by oocyte (can be tested by seeing if partner sperm can fert donor eggs)

257
Q

SHould ICSI be re-performed on oocytes that have already been inseminated?

A

NO

258
Q

oocyte activation (CAUSED BY FEERTILIZATION- gamete fusion)

A

sperm-specific protein, phospholipase-C zeta (PLCzeta) is pivotal for oocyte activation

After gamete fusion, oscillation waves of Ca2+ released from oocyte- these spikes modulate cortical granule exocytosis, release meiotic arrest, regulate gene expression, recruit maternal mRNA, initiate embryo genesis

requires extracellular Ca2+ to replenish cellular stores

Can be induced by action of sperm injection- bypasses AR.

259
Q

Abnormal PLCzeta results in

A

male infertility

abnormal PLCzeta structe impairs function in human sperm - cannot properly cause oocyte activation

260
Q

Artificial oocyte activation

A

exposure to Ca2+ ionophore can be applied immediately after ICSI (15 min conc 10umol/L)

Can be used with male pateints sperm with globozoospermia (no acrosome)

261
Q

Mature oocytes have how long to be fertilized post-maturation?

A

12 h.
mature oocytes have a limited lifespan in regard to developmental competence, following ovulation/polar body extrusion/egg retrieval

262
Q

Why might motility be the best selector for sperm for ICSI?

A

the embryo derives its centriolar apparatus and mitotic potential from the sperm, the use of sperm with a damaged or abnormal centriole could result in embryos with severe cleavage arrest or irregular cleavage patterns

motility usually = intact centrosomes&raquo_space;important for mitotic divisions

263
Q

which test can be used prior to ICSI to determine live sperm for use?

A

hypo-osmotic swelling test

not a live/dead stain bc would kill the sperm (cant inject a cell with stain)

264
Q

Physiological ICSI (PICSI)

A

asses sperms ability to bind to hyaluronan on the surface of an ICSI dish. THose sperm that can bind are used for ICSI

(used to select sperm with lower levels of DNA frag and against those with poor morphology, high levels of aneuploidy in sperm)

265
Q

Quality Management (QM) program includes:

A
  1. Quality Control (QC)
  2. Quality Assurance (QA)
  3. Quality Improvement (QI)

each defined by CLIA 1988

266
Q

Quality Control (QC)

A

inspects all aspects of a system, to ascertain that all equipment and processes are functioning as required (daily, weekly checks)

267
Q

Quality Assurance (QA)

A

verification and validation that QC data are analyzed so that errors and problems are accurately detected and addressed (when baselines, threshold limits are set, monitoring lab personnel assessments, KPIs met etc)

268
Q

Quality Improvement (QI)

A

Setting benchmarks

269
Q

WHat would the most sensitive MEA testing be

A

one-cell outbred (wildtype) mouse embryos using serum (protein)-free medium.

270
Q

Why are sperm cells ideal to freeze

A

high SA: vol ration
little cytoplasm
contain less intracellular water than other cells
exist individually so easy dehydration

271
Q

What are the three non physiological conditions that cells must tolerate during cryopreservation?

A
  1. exposure to multimolar concentrations of CPAs
  2. cooling to subzero temps
  3. removal of almost all cell water or it’s conversion to a solid state
272
Q

permeable CPAs are characterized by their:

A
  • relatively low molecular weight
  • high solubility in water
  • cell permeability properties
  • non-toxic properties at high intracellular concentrations
273
Q

what is the most common CPA for freezing sperm?

A

glycerol

274
Q

The principles of cryopreservation are based on the laws of

A

Thermodynamics

275
Q

What happens if CPAs are not added during sperm freezing?

A

cryodamage due to plasma membrane swelling as water expands + acrosomal breakdown

276
Q

Glycerol as a CPA works in what way?

A

reduces the water content of sperm cell to minimize intracellular ice formation

277
Q

when sperm are placed in CPAs to the expand or shrink initially

A

Shrink, due to reduction of intracellular water due to increased extracellular osmolarity

278
Q

when is osmotic equilibrium reached after addition of CPA

A

when CPA penetrates the cell and displaces intracellular water and sperm cell returns to it’s original volume

279
Q

addtion of CPA is accompanied by?

A

reduction in temperature. during the cooling process, when -5 – -15C reached, extracellular ice formation occurs and induces the development of extracellular solid phase

280
Q

addition of CPA is usually accompanied by

A

reduction in temperature.

when temp reaches -5 to -15C, extracellular ice formation induces solid phase

281
Q

when temp reaches -5 to -15C, extracellular ice formation induces solid phase, the inside of the sperm remains?

A

unfrozen but super cooled.

Supercooled intracellular water has a higher chemical potential and diffuses out of the cell osmotically. (water still diffusing out at this point)

282
Q

As temp drops below -15C, freezing continues extracellularly resulting in

A

Hypertonicity (less water outside than in so pushes water out), removing more water from sperm cell resulting in almost complete dehydration

283
Q

What can cause issues during dehydration of sperm cells?

A

Not allowing enough time for dehydration and osmotic equilibrium

284
Q

Same process for thawing and freezing in regard to time

A

slow in- slow out (room temp for 30-60 min)

fast in- fast out (37C bath for 5-10min)

285
Q

Do vitrification CPAs have high or low molecular weight?

A

high

286
Q

what is the common concentration of glycerol used for freezing human sperm?

A

5-10% volume to volume (v/v)

287
Q

TES and TRIS are

A

extenders containing zwitterions as buffers, sodium citrate and egg yolk in addition to glycerol are considered the optimum extender

288
Q

sperm extenders are

A

liquid diluent added to semen to act as a buffer to protect sperm from their own toxic byproducts, cold shock, osmotic shock during cryo

Zwitterions are best- bind free hydrogen and hydroxyl ions in media

289
Q

egg yolk in extenders acts to

A

maintain sperm viability by improving sperm membrane fluidity

290
Q

What is the glycerol concentration of TEST yolk buffer?

A

12% glycerol - duluted with sperm suspension 1:1 v/v sp overall glycerol concentration in specimen is 6%

291
Q

Why use an egg yolk free buffer?

A

avoid potential allergic reactions and reduced exposure risk to animal products

292
Q

What is the method for slow freezing human sperm?

A

Usually, CPA medium is added slowly drop wise to sperm suspension (1:1).

slow cooled for 10-30 min before plunging in liquid nitrogen

293
Q

When sperm samples are slow cooled in vapor prior to plunge in LN2, what dictates the cooling rate?

A

relatively uncontrolled cooling rate- dictated by distance of samples above the surface of the LN2.

294
Q

When sperm samples are slow cooled in a programmable freezer prior to plunge in LN2, what dictates the cooling rate?

A

automated system using a cooling rate of:

-0.5C/min from room temp. to -5C

freezing rate: -10C/min from -5C to -80C or -90C

then LN2

295
Q

Sperm vitrification

A

not found to be any more successful than slow freeze methods- not commonly used.

Sperm are sensitive to high concentrations of CPAs used for vit, and causes sperm membrane damage and increased DNA damage

296
Q

Freezing small numbers of sperm, say from surgically retrieved methods require additional methods

A

poor sperm quality = poor sperm cryoviability

biological carriers: denuded mouse zonae used as a holding container, or vitrification in microdrops using non-biological devices - technically complex methods

297
Q

Donor sperm cryopreservation, storage and distribution from directed and annon. donors are governed by:

A

states and FDA through the federal gov.

(example special New York law: donor sperm that resulted in pregnancy must be kept for at least 25 years after semen distribution)

298
Q

WHo establishes the standards for donor sperm use?

A

American Association of Tissue Banks (AATB)

299
Q

Who establishes the guidelines for donor sperm use?

A

American Society for Reproductive Medicine (ASRM)

300
Q

Who do sperm banks freeze specimens for?

A

anonymous donors, directed donors, and client depositors

301
Q

All sperm donors must undergo what requirements before being eligible to donate sperm?

A

intensive screening procedures including:
-Comprehensive application- medical, social, sexual history
-family medical and genetic history at least 2 generations
-Negative tests for infectious diseases and STIs
-pass a physical examination looking for evidence of disease
-medical history exam looking for high-risk behavior

302
Q

Requirements for anonymous donor semen

A

frozen and quarantined for at least 6 months prior to use.

after this 6 mo. and prior to release of specimens for use, donor must be retested for relevant infectious diseases.

All donors must sign a consent for their sperm to be used.

303
Q

Anonymous donor

A

a semen donor whose identity is unknown to the recipient

304
Q

Client depositor

A

individual who banks his semen for deffered insemination of SIP or long term storage. (he is not a donor at the time of semen cryo)

305
Q

Semen donor

A

Individual who provides semen for cryobanking and use in a recipient other than his SIP. Can be annon or directed

306
Q

Directed donor

A

donor who is personally known to the recipient and who directs his semen for use by a particular recipient

307
Q

Semen bank

A

tissue bank that collects, processes and stores and/or distributes human sperm for use in ART procedure including IUI and IVF.

308
Q

Examples of syndromes that can happen if you fertilize with aneuploid sperm?

A

Klinefelters Syndrome (XXy-XXXY), Turner Syndrome (XO), or Down Syndrome (trisom 21)

Gain or loss of whole chromosome

309
Q

3 main types of chromosomal abnormalities

A
  1. Gain/loss entire chromosome
  2. translocations
  3. deletions and microdeletions
310
Q

Nondisjunction

A

failure of homologous chromosomes to disjoin during metaphase of meiosis (sister chromosomes don’ split)

311
Q

Klinefelters syndrome

A

1:500 births
Sex chromosome abnormality
extra 1 or multiple X chromosome
XXY, XXXY, XXXXY
azoospermic with small atropic testis

312
Q

XYY males

A

usually fertile

313
Q

XX males

A

genotypic females and phenotypic males
dev. of testis and male repro tract with masculinization but atrophic testis (Azoospermia)
Partial chromosome defects (part of a Y chromosome with SRY gene)

314
Q

Partial chromosome abnormalities (translocations and deletions)

A

during chromosome crossing-over, sections of chromosomes may recombine to the wrong chromosome (translocation) or be lost (deletions)
example: Robertsonian Translocation (most common) long arm with a long are and small with a small(misalignment during meiosis)

Most carriers of these translocations are phenotypically normal until trying to reproduce

315
Q

Y chromosome microdeletions

A

azoospemic men found to have microdeletions on Y chromosome at position Yq11 (encodes AZF azoospermic factor region). Usually identified by a multiplex PCR assay
(dont make offspring if Y microdeletion found–offspring may have as well)

316
Q

Copy number variations (CNV)

A

usually identified by assay, array comparatively genomic hybridization (aCGH) -oligonucleotides on glass slide and patient and control samples fluorescently labeled to show a loss or gain of copy number. this microarray can recognize unbalanced gains or losses but not translocations

317
Q

How are autosomal gene mutations found?

A

DNA sequence analysis (sanger sequencing); can identify point mutations

318
Q

Next Gen sequencing

A

high throughput sequencing

multiple short reads sequenced in parallel

Improved accuracy and speed compared to Sanger sequencing

319
Q

Whole genome sequencing

A

uses Next Gen seq to sequence EVERY base pair

Whole exome sequencing - uses next gen seq to look at only to he coding portion of genome (more cost effective and can identify up to 85% of mutations that are disease impacting)

320
Q

How can XX females or XY males have incorrect repro organs?

A

Small piece of y chromosome containing SRY in female, or disfunctional SRY or SOX9 gene in male

321
Q

Hypospadias

A

urethral opening not at tip of glands penis, posterior somewhere

322
Q

Cryptorchidism

A

failure of testicular descent (3% males born) over 2500 genes could be potentially involved

Even if testicle descends in infancy, continued impact on spermatogenesis possible and increased cancer risks

323
Q

Kallman-Syndrome (Hypogonadotropic Hypogonadism

A

failure of GnRH releasing neurons to migrate to olfactory lobe during fetal development (X-linked disorder)

324
Q

Androgen insensitivty syndrome

A

Androgen receptor gene defects - inactivation of the androgen receptor (female external genitalia and testis present internally )

325
Q

high resolution banding cytogenetics

A

karyotyping - visualizing chromosomes using fluorescent probes

326
Q

FISH

A

sperm fluorescent In situ hybridization

fluorescently labeled probes to chromosomes 13,18,21 X and Y are hybridized to the sperm (these chromosomes are the only ones that if they make an embryo, can result in a viable pregnancy)

327
Q

big or small sperm head

A

higher chance of chromosomal abnormality

328
Q

males with Y chromosome micro deletions

A

should not be candidates for TESE as male embryos will have the same mutation

329
Q

Sperm may be found in XXY males (TESE) but never XX males

A
330
Q

PLCzeta

A

induces oocyte activation

delivered by sperm into oocyte after sperm enter oolema

Exam Qs (1 decks)

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