Pregnancy Obtained with Human Testicular ... - Wiley Online Library

Journal Copyright

of Andrology, Vol. 15,

1994

Supplement

Society of Andrology

© American

Pregnancy Spermatozoa

Obtained with Human Testicular in an In Vitro Fertilization Program

R. SCHOYSMAN, P. VANDERZWALMEN, L. GEERTS, E. VAN ROOSENDAAL, From the Schoysman 42, 1800 Vilvoorde,

Infertility Belgium.

M. NUS, L. SEGAL, A. SCHOYSMAN-DEBOECK

AND

Management

Foundation,

van Helmont

ABSTRACT: The fertilizing capacity of human testicular spermatozoa and the positive outcome of an in vitro fertilization program open a wide range of opportunities for men suffeting from obstructive and Inoperable azoospermia. In six cases, subzonal sperm injection or Intracytoplasmic sperm injection techniques were applied to Inject testicular spermatozoa into human oocytes. The fertilization rate after testicular sperm Injection reached 45%. Normal cleavage was

p

atients

with

complete

agenesis

of the

vitro fertilization

(IVF)

ididymal

aspiration have

sperm

though pregnancies ber, 1984; Temple-Smith et al,

1988;

vas

deferens

are routinely program, where

obstruction

(MESA)

epAl-

of the

maturation

Unfortunately program

had

could

process.

patients entering the MESA because no spermatozoa Considering the drop in the fertiliza-

to be disappointed

be retrieved.

ejaculated, seemed in sperm

Yet the fertilizing been demonstrated

ability

Yanagida, rayama

1991),

the

hamster

deferential, and epididymal unlikely fertilization capacity retrieved from the testis itself. of testicular spermatozoa rabbit (Brackett et a!,

(Uehara,

1977),

et a!,

1993). It was therefore strategy for patients with failed

ultimate collection

was

isolate

to perform

spermatozoa

Correspondence Unit for Infertility, voorde,

Brussels,

in order

to: Dr. Robert Avenue Rene

a biopsy to inject

Schoysman,

Comhaire,

and

boar

decided

them

the

sperm

testis either

and into

April

to the

van Helmont Hospital, 69, 1080 Bruxelles, Vil-

19, 1994; accepted

for publication

and replacement

of 10 embryos in 6 patients resulted in and one ongoing pregnancy. Human sperm, in vitro fertilization, subzonal sperm

pregnancy,

perivitelline

the

space

or directly

into

microsurgical

cytoplasm

of the

oocytes.

been performed, the specimen was simultaneously collected and Bouin-flxed for histologic evaluation. The location of the biopsy was equatorial and not in the neighborhood of the rete testis. The sample was delivered to the biologist in a petri dish containing Earle’s medium supplemented with 7.5% cord serum. It was divided into fragments of tissue of 1 mm in diameter. These were then dissected under the microscope where one can see the tubules progressively diffuse their contents into the medium (Fig. 1). The whole procedure results in a fluid suspension packed with numerous spermatogenic cells and spermatozoa. Some of these spermatozoa exhibited sluggish motility. The suspension was then centrifuged for 10 minutes at 300 x g on a mini-percoll gradient and incubated in Earle’s medium at room temperature until microinsemination. An average of 150,000-300,000 spermatozoa was usually obtained.

(Ha-

that

epididymal of the

has 1978;

Belgium.

for publication 26, 1994.

Received

tember

in

Vaartstraat,

This preliminary study concerns six excretory azoospermic men who were referred because of a congenital absence of the vas (A-D, F) or failed vasoepididymostomy (E). In the first group, the fertilizing capacity of human testicular sperm was evaluated in three patients (A-C) entering the MESA program, along with a positive epididymal sperm aspiration. In the second group (D-.F), there was a complete absence of spermatozoa in the epididymis even after several levels were explored. Considering this to be unsolvable, a testicular biopsy specimen was collected. A routine testicular biopsy was performed in which a sample of 4 x 4 mm was collected. If no previous diagnostic biopsy had

1990;

7% of our

tion rate between sperm, it always would be observed

Laboratory,

Materials and Methods

Olar et al, 1990; Cognat and Guerin, 1991), it is generally accepted that the fertilization capacity of spermatozoa at the epididymal level is reduced, probably because of a bypass

I. VF.

injection, intracytoplasmic sperm injection, epididymal sperm aspiration. J Androl 1994;15:IOS-13S

partners’

after MESA (SilPryor, 1987; Silber et al,

Ziekenhuis,

one chemical Key words:

a

performed.

et al, 1985; et al, 1989; Jequier

Schoysman

observed

in our in

microsurgical is

reported

been

or with

accepted

G. SEGAL-BERTIN,

Sep-

los

Schoysman et al

.

Pregnancy

Obtained

with Testicular

FIG. 1. The testicular biopsy Is dIvided into small samples cell suspension is obtained.

In the case of subzonal

insemination

(SUZI),

Sperm

11$

are gently crushed between two slides until a homogenous

of 1 mm3. These small fragments

the spermatozoa

were incubated in 0.1% pentoxyfilline and 50% follicular fluid (Fournier-Delpech and Thibault, 1991). When intracytoplasmic sperm insemination (ICSI) was used, the sperm suspension was incubated in 5 mM CaC12 and centrifuged for 5 minutes at 1,800 x g before injection into the cytoplasm. The ovarian stimulation was started with a short analogue protocol: Suprefact Hoechst (buseriline acetate) nasal spray was given on day I of the cycle and applied four times daily, and human menopausal gonadotropin (Pergonal Serono 75 U), 150 U daily, was given beginning on day 3 of the cycle. Follicle puncture took place 34 hours after injection of 5,000 IU of human chorionic gonadotropin (HCG). Oocytes were collected and placed in culture in Earle’s medium supplemented with 7.5% cord serum. After oocyte retrieval, cumulus cells and corona radiata were removed by incubating the oocytes into Earle’s medium with 160 IU hyaluronidase for up to 1 minute. Only intact oocytes that had extruded the first polar body were microinseminated with the SUZI (Ng, 1988) or ICSI procedure (Palermo et a!, 1992). All microinseminations were performed in prewarmed plastic petri dishes under oil. The oocyte was immobilized by a holding pipette. For SUZI, up to 10 spermatozoa were injected into the penvitelline space. In the case of ICSI, the microinjection pipette was pushed equatorially through the zona pellucida and the cytoplasm. After injection of one testicular spermatozoon, the pipette was gently withdrawn and the oocyte released.

Results Testicular Sperm Characteristics Outcome of Insemination

and

Morphologic

included

very

low

analysis number

patients (A-C), were fertilized with

was

of sperm

5 out with

epididymal

sperm

(Table

1. Outcome of fertilization spermatozoa in six patients

A1/2 B C Total

Fertilization with epididymal sperm

SUZI

SUZI

0/2

1/4

5/11(45%)

D E F Total

with testicular

Fertilization

3/3

the

group

ICSI

D-F

and/or epididymal

Embryos Transferred

Frozen

5/6

2

3

4/5

3

4

0/3

where

Pregnancy

1

No No No

1 1 2

NO Chemical Ongoing

9/14 (64%) 1/9 1/8 2/7

4124

first

1). In patients

with testicular sperm ICSI

considering In the

of 11(45%) metaphase II oocytes testicular sperm and 9 of 14 (64%)

Table

Patients

not

available.

(17%)

of

Journal of Andrology sperm

were

was

17%

SUZI

found

only

(4/24).

in the

testis,

Fertilization

as with

ICSI.

the

was

After

24

fertilization

observed

hours

rate

as well

of culture,

with

embryo

quality was evaluated and showed to be comparable to that in our routine IVF. All six patients received testicular embryos ical and

during transfer, which one ongoing pregnancy.

pregnancy,

two

of seven

oocytes

with

testicular sperm showed two pronuclei. After 44 hours, a 4-cell grade A embryo and a 2-cell grade B embryo were

transferred.

fertilizing capacity without A further problem is the seminated

programs.

of fertilization

sperm (Schoysman had never before

of oocytes

et al, 1 993a,b) been reported

stressed that although fertilization the animal model, cleavage was

could arrested

physiology

pathway

is of proven

flexibility,

with testicular bypass of the

spermatozoa epididymal

curred. firmed

Fertilization with testicular by Craft (1993), Tournay and

Hirsch

classical IVF Physiology considerable important the

(1993)

who

but

amount part this

numerous

Epididymal

the

pregnancy

is surprising environment sperm (personal

observed

insemination. of the epididymis

has been concommunicafertilization

has

been

target

of a the by

secreted

the

by the

flexibility flexibility

have in the became

demonstrated

need for apparent

epididymal

of vasoepididymostomies performed at different sense, were epididymal

because levels of the

is one

more

of

maturation. This analysis of results

such operations epididymis and,

challenge

one must

about

fertilizing

capacity

of testicular

cause

the

microinsemination

in obtaining

to the

although

preceding cytoplasm

oocyte.

to the At

in-

surface

the

present

look

forward

to comprehensive

a clinical future situations

number questions

point of view, there for such an approach. in which

although

data

of attempts in that arise.

is undoubtedly an There are actually

the epididymis

does

spermatogenesis

not

contain

is normal,

both

If further work with testicular

obstructions. of fertilization

spermatozoa, an important breakthrough for the handling of these cases could be achieved. Finally, fertilization with testicular sperm may threaten the

microsurgeon.

gynecology inated by epididymal way and pling

When

one

considers

to what

extent

tubal microsurgery has been practically IVF, one may wonder whether in the microsurgery will not be replaced by a simple

or puncture

and

an oocyte

follow the same testicular biopsy

collection

in

elimfuture, pathsam-

by echographic

pick-up.

Conclusion Fertilization

are in a

by testicular

numerous

ongoing

andrological

pregnancies

crosurgery even when spermatozoa

cannot

sperm cases.

are possible. solve

all types

offers

new

possibilities

Fertilization Because

as well epididymal

of excretory

azoospermia

normal spermatogenesis is present, collected from testicular tissue

to be a valuable Further work

approach. in the human

is at hand

as mi-

the may for

use of prove

collecting

data on sperm recovery from testicular tissue and comparing the quality and quantity of testicular spermatozoa with the score from biopsy reading.

References spermatozoa

physiology,

the steps into the

the

bind

are clearly in contradiction with of the importance of epididymal on motility and fertilizing capacity

of spermatozoa. The use of testicular ization

degree

experiments demonstrating the importance of function in humans (Schoysman and Bedford,

1986). Such approaches the established concept transit and its influence

ymal

We eagerly

in congenital or postinfectious improves the success rate

for

1986; of clin-

an apparent

epididymal during the

fertilize

can

bypass. subzonally

after

of experimental work stressing organ plays in sperm maturation

glycoproteins

situations

obbecause has oc-

epithelium (Bedford et al, 1973; Orgebin-Christ, Fournier-Delpech and Thibault, 1991). A number ical

in em-

observed. Neither follow what is con-

of maturation.

tained a total

tion),

be obtained at an early

and

spermatozoa

testicular

followed by pregnancy in humans. It is to be

bryo stage and no implantations were testicular nor epididymal spermatozoa normal

with

spermatozoa

insemi-

can exhibit

microinsemination fact that even

different centers on a larger to unravel further the many

many

the

ooplasm

From important

observation

sidered

testicular

IVF

1994

time the results obtained do not offer clearcut data on this important topic. We can only hope that other groups show interest in working with testicular spermatozoa in IVF from order

Discussion The

by Hirsch (1993) using classical do prove that testicular spermatozoa

of the

resulted in one biochemIn the case of the ongoing

microinseminated

obtained nation

Supplement

concepts

of epidid-

be careful

in speaking

spermatozoa

procedure

the penetration of the oocyte.

fertil-

bypasses

of the Still the

beall

spermatozoa fertilizations

of

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