Micromanipulation as a Clinical Tool ppt

14

Micromanipulation as a Clinical Tool

Jacques Cohen

Tyho-Galileo Research Laboratories and Reprogenetics, West Orange,

New Jersey, U.S.A.

INTRODUCTION

Micromanipulation involves a well-integrated set of technologies in assisted

reproductive technology (ART). Its applications are diagnostic as well as

therapeutic, and it is practiced in mature gametes and all stages of preimplan￾tation embryos. It is used in biopsy for preimplantation genetic diagnosis

(PGD), intra-cytoplasmic sperm injection (ICSI), assisted hatching, and in

other more controversial areas such as egg freezing via zygote reconstitution,

cryopreservation of isolated testicular spermatozoa, and cytoplasmic or

mitochondrial transfer for reversal of cytoplasmic and potentially nuclear

incompetence (1–6). In this context, it becomes increasingly difficult to dis￾cuss micromanipulation as a separate subject. In fact, the need for a separate

assessment appears almost artificial.

When different fields merge in science, exciting developments can be

expected. This has occurred numerous times in ART, first in the integration

of biochemistry and reproductive endocrinology, and later when cryobiol￾ogy and applied genetics emerged as tools to improve efficiency and safety.

No reproductive specialist could have predicted that the field of experi￾mental micromanipulation would have such an enormous impact on assisted

reproduction less than two decades after the first relatively simple but

elegant applications appeared (1–3). Since then, hundreds of thousands of

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babies have been born worldwide from micromanipulative methods aimed

at alleviating male infertility and enhancing implantation and the exclusion

of chromosomal and single gene disorders. Some laboratories now have

three or more complete stations for micromanipulation. Rather than having

some embryologists sub-specialize in the area of micromanipulation, the

practice in some laboratories, most embryologists now aim to become pro￾ficient in one or more micromanipulation techniques.

The main emphasis here will not be already integrated procedures such

as ICSI and in part embryo and polar body biopsy, which are covered in

depth by other chapters of this book, but on other innovative techniques.

The different procedures and concepts will be discussed in two sections;

the first will focus on gamete micromanipulation and the second will deal

with the manipulation of embryos. It is also important to note that the

topics discussed in this chapter are often considered controversial or unpro￾ven and are seen by some as hazardous to clinical care and even the human

species at large. No review would be complete if it does not refer to the alter￾native opinion. A recent 2004 opinion by Cummins gives a very different

perspective of some of the technologies described below (7).

GAMETE MICROMANIPULATION

Cryopreservation of Spermatozoa Under Zonae

Men who are azoospermic can be successfully treated through surgical iso￾lation of spermatozoa from their testicles or reproductive tract (8–10).

Repeated surgical procedures, however, may not only be costly but invasive,

especially in the case of testicular sperm extraction (11). Repetition can, in

some cases, be avoided by normal cryopreservation of spermatozoa, but

only when sufficient numbers of functional cells are isolated (12,13) Freezing

methods are now available that can freeze and recover very few or even sin￾gle spermatozoa, and avoid the need for repeated surgical sperm extraction

(4,14,15). Single sperm can be frozen by insertion of spermatozoa into ani￾mal or human evacuated zonae pellucidae or through variations of this

method such as freezing in cryoloops.

Recovery rates of spermatozoa frozen and thawed in evacuated

human or animal zonae are high, with motility recovery rates in excess of

75% (4,15). In standard freezing protocols, centrifugation is essential. But

in this protocol, washing can be accomplished by individually pipetting

and removing the cryoprotectant.

In addition to reducing the need for repeated sperm retrievals and

perhaps donor sperm, this approach has the advantage of avoiding the

uncertain outcome of surgical extraction by freezing spermatozoa and

retrieving eggs at different times (4,16). The time-consuming search for sper￾matozoa can be conducted independent of an egg retrieval.

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Details of Procedure and Choice

of Technical Details

Micromanipulation can be performed using polyvinyl pyrrolidone (PVP) as

a tool to slow the insertion of spermatozoa into the zonae and to withdraw

spermatozoa from the zonae after thawing. Two different solutions of PVP

are recommended: (i) an 8 to 10% solution for sperm capture and insertion

into empty zonae (this is produced by a number of manufacturers with vary￾ing results) and (ii) a 10 to 12% solution for sperm recovery from the thawed

zonae. The ICSI procedures using thawed spermatozoa should be per￾formed at 37
C, but all other micromanipulations can be performed at room

temperature in order to reduce sperm velocity and perhaps prolong survival.

The microtools needed for zona opening, avoidance of zona collapse, cell

extraction, and ICSI have been described (3,4).

The pilot experiments were conducted using spermatozoa from surgi￾cal retrievals and spermatozoa not cryopreserved from men with normal

semen analysis to test the fertilizing ability of donated research oocytes by

ICSI. Human-evacuated zonae can be obtained from multiple sources:

immature eggs, unfertilized ICSI eggs, and abnormal embryos that were

not exposed to sperm suspensions.

It was shown that two small incisions in the zonae improved extraction

of the egg ooplasm and insertion of the sperm cells into the evacuated zonae

by preventing the collapse of the zona during suction and excessive inflation.

With gained experience, a single-hole technique may be effective as well. At

first, holes were made chemically in some pre-fertilization zonae by releasing

acidified Tyrode’s solution from a 10 mm open microneedle. However, pro￾gressively motile spermatozoa escaped, resulting in poor recovery rates. This

can be avoided by cutting a hole in the zona mechanically using partial zona

dissection with a spear-shaped closed microneedle. Alternatively, one can

use a laser-mediated opening of the zona pellucida, but the efficiency of this

needs to be shown in clinical trials (17).

Cytoplasm can be extracted using a larger micropipette, connected in

turn to a suction device. The zona is positioned so that one of the two inci￾sions is at the 3 o’clock position. The beveled microtool is inserted through

the aperture using the sharp edge on the lower end. The tool is moved

through the oolemma, and the cytoplasm is fully aspirated until the zona

is empty. The pipette is occasionally emptied outside the zonae as more

medium is sucked up to remove any sticky cytoplasm from the pipette tip.

Mouse and hamster zonae as well as human eggs and embryos can also

be prepared in the same fashion.

Spermatozoa can be released into the 10% PVP solution prior to inser￾tion into empty zonae. They are individually taken from small 2- to 5-mL

droplets of sperm suspension using an ICSI microtool. Spermatozoa can

be injected into the empty zona while motile or can be immobilized before

Micromanipulation as a Clinical Tool 285