Sample preparation for ART

2. Male Infertility
•About 25% of all infertility
•First IUI done in year1700 --UK
•assisted reproduction, a quarter of a century ago,
required the isolation of motile spermatozoa

3. 1969-1st sperm separation methods -one or two washing
(media) procedures with subsequent resuspension
(media)of the male germ cells (1-3)
1. Edwards RG, Bavister BD, Steptoe PC: Early stages of fertilization in vitro of human
oocytes matured in vitro. Nature 1969
2. Edwards RG, Steptoe PC, Purdy JM: Establishing full term human pregnancies using
cleaving embryos grown in vitro. Br J Obstet Gynaecol 1980
3. Lopata A, Brown JB, Leeton JF, Talbot JM, Wood C: In vitro fertilization of
preovulatory oocytes and embryo transfer in infertile patients treated with
clomiphene and human chorionic gonadotropin. Fertil Steril 1978

4. 1984-a single wash - followed by a swim-up
procedure from the cell pellet.
Later on more sophisticated methods were
developed to obtain sufficient amounts of motile,
functionally competent spermatozoa for IUI.
Mahadevan M, Baker G: Assessment and preparation of semen for in vitro fertilization.
In: Clinical In Vitro Fertilization Edited by:Wood C, Trounson A. Springer-Verlag, Berlin; 1984

5.  Swim-up (Mahadevan and Baker, 1984)
 Percoll density gradient centrifugation (Gorus &
Pipeleers, 1981)
 Glass-wool (Paulson & Polakoski, 1977)
 Sephadex bead filtration (Lopez et al.,1993)
PRINCIPLE & PREPARATION TECHNIQUES
Are based upon Migration, Density Gradient
Centrifugation and Filtration

6. The ideal sperm separation technique
should
 be quick,
 easy and cost-effective,
 isolate as much motile spermatozoa as possible,
 not cause sperm damage or nonphysiological alterations of
the separated sperm cells,
 eliminate dead spermatozoa and other cells, including
leukocytes and bacterias,
 eliminate toxic or bioactive substances like decapacitation
factors or reactive oxygen species (ROS) and,
 allow processing of larger volumes of ejaculates.

7. The quality of sperm samples is one of the factors
determining successful IUI
(Ombelet et al. 2003)

8. Objective
 Concentrate the motile spermatozoa in a fraction -free
of seminal plasma and debris
 Maximam the changes of fertilization to provide as
many normally fertilized ooctes as possible
 Elimination of seminal PG, lymphokines,
cytokines and infectious agents
 Reduce the number of free oxygen radicals

9.  The male partner should collect semen into a sterile,
clearly labelled disposible plastic jar with in a room
adjecent to the IUI laboratory
 The time of sample collection should be recorded on the
label
 Semen should be prepared soon after liquefaction
 If liquefaction delayed or specimen viscous; mixing the
specimen 1:1 with medium may help or enzymatically
liquefaction can be done (ie; α-amylase, hyolurinidase,
tripsin based dissolving sol.)
 10 μl of the sample is taken to check sperm consentration
and motility
Collection of sperm

10.  Initial assesment of density and motility--- choice
most appopriate method of sperm preparation
 The choice of sperm preparation method depens on,
the motile count,
ratio between motile/immotile count,
 volume,
 presence of antibodies,
 agglutination
Sperm Preparation

11. WHO Lab Manual of Human Semen 2010
5th centile 95%
CI
Volume 1.5ml (1.4-1.7)
P H 7.2
Motility 40% (38-42)
Progressive
Non progressive
Immotile
32% (31-34)
Vitality
(intact membrane)
58% (55-63)
Count 15X106 12-16X10 6
Normal forms 40% (30-4)
10/24/2023 11
The lower reference limit for semen analysis

12. 10/24/2023 12
A cut off level of:
10million motile spermatozoa after washing .
Metaanalysis (Van Weert et al 2004 )
30-50% Total sperm motility before sperm preparation
( Ombelet W et al 1996
Dickey et al 1999, Montanaro et al
2001, Lee et al, 2002)

13. Swim-up
 Used largely in IUI laboratories around the world.
 swim-up --standard technique for patients with
normozoospermia and female infertility.
This procedure has several variations

14. Standart swim-up
The methodology of conventional swim-up is
based
on the active movement of spermatozoa from the
prewashed cell pellet into an overlaying medium.
Typically, the incubation time is 60 minutes.

15. Direct swim-up from liquefied semen
 A maximum recovery is obtained by using multiple
tubes with small volumes of semen per tube, thus
maximizing the combined total interface area between
semen and culture medium.
 Mortimer suggested the use of 250 μl semen and 500
to 600 μl culture medium per tube

17. Pellet and swim-up
 Alternatively, semen sample may be diluted and
centrifuged and pellet overlaid with medium
 Useful for viscous samples
 Not recommended when motility is very poor or large
degree of cellular contamination and debris
 Has disadvantage of peroxidative damage during
centrifugation with defective sperm and white cells*
*Aitken RJ, 1990

18. Density gradients
 Various gradient procedures
 It is rapid, relatively simple to perform
 Abnormal sperm, immotile sperm and debris can
largely eliminate
 Generally the recovery of motile sperm is greater with
gradients but the percentages of sperm with
progressive motility is usually lower than with swim-
up

19. Density gradients
 Colloidal silica particles (coated with PVP:Percoll)
 Silane coated silica particles (Isolate, PureSperm,
SilSelect)
 Nycodenz (iodinated hydrocarbon),

20. The ejaculate is placed on top of the density media
with higher density and is then centrifuged for 15–30
minutes
Highly motile spermatozoa move actively in the direction
of the sedimentation gradient and can therefore penetrate
the boundary quicker than poorly motile or immotile cells,
thus, highly motile sperm cells are enriched in the soft
pellet at the bottom

21. Density gradients
 Density gradients protect the sperm from trauma of
centrifugation
 High proportion of functional sperm can be recovere
 Two or three step gradients are simple and highly
effective in preparing motile sperm from suboptimal
samples
 In general longer centrifugation time increases the
recovery of both motile and immotile sperm
 Debris, round cells, abnormal forms never reach the
bottom of the tube because of their low density

22. Density gradients
 Gradients with larger volumes result in improved
filtration, but decreased yield
 Three layers of mini-gradient improve filtration,
recovery of sperm from severely oligospermic samples
 Samples wiht large amont of debris should be
distributed in smaller volumes over several gradients
 Severely asthenoozospermic samples,with normal
density can also be distrubuted over a series of mini-
gradients

23. Density gradients

24. Which procedure for IUI?
Swim-up/Gradient?
Boomsma CM, Heineman MJ, Cohlen BJ, Farquhar C .Cochrane Reviews March 2004

25. Which procedure for IUI?
Swim-up/Gradient?
Cochrane Database of Systematic Reviews, 2004

26. Semen preparation techniques for intrauterine insemination
Boomsma CM, Heineman MJ, Cohlen BJ, Farquhar C
 Summary
 review found that there is insufficient evidence to recommend
any specific sperm preparation technique for subfertile couples
undergoing intrauterine insemination (a procedure which places
sperm directly into the uterus) as there were no differences in
pregnancy rates. More research is needed.
Cochrane Reviews March 2004

27. Sperm processing
 Samples with an acceptable number of motile
sperm ( > 20 millions / ml ) can be processed
efficiently by sperm wash twice and swim-up.
 Poor quality semen samples should be processed
using density gradient centrifugation DGC.
Morshedi M et al, 2003

28. 10/24/2023 28
•Easy to perform
•Training is easy
•Less invasive
•Risks are minimal
•Quality control possible
•Costs are minimal
Advantage of IUI

29. Complications
 Relatively low success rate / cycle.
 PID 0.01-0.2%.*
 MP
 Prematurity & low birth weight.**
* Dodson and Haney, 1991
*Ombelet et al 1995
**Wong et al 2002, Gaudoin et al 2003, Ombelet et al 2006.

30.  THANK YOU