Paper Ginekologi

Preparasi Sperma pada Inseminasi Intrauterin (IUI)

Pembimbing :
Dr. H.M. Haidir, Sp.OG
Oleh :
Meiliska Aulyanissa
NPM. 1030230

Definisi
Teknik bantuan reproduksi dengan cara memasukkan secara
langsung spermatozoa yang bergerak ke dalam kavum uteri
pada waktu yang tepat dari siklus menstruasi pasien, dimana
sebelumnya telah dilakukan preparasi terhadap sperma.

Indikasi Inseminasi Intrauterin (IUI)

Menurut Sahakyan (1999), indikasi IUI adalah sebagai berikut :
Indikasi

Jumlah

Pasien

n Siklus Fekunditas

(%)
Faktor Pria

32 (11,7)

Anovulasi

73 (26,6)

13

Endometriosis

55 (20,1)

12

Unexplained

97 (35,4)

10

Faktor Tuba

16 (6,2)

Patient Selection Criteria

for IUI
Female
age <40 years
Minimum of 1.5 years of infertility
Patent fallopian tubes confirmed by laparoscopy / hysterosalpingogram
Presumptive proof of ovulatory cycle
Ultrasound evidence of mature follicles & ovulation
Luteal phase progesterone (P) cutoff >35 nmol/L

Male partner:
Two semen analysis revealing at least 10 million recovered motile sperm /
whole sample

Keuntungan

1. Sperma >> cavum uteri

2. Jarak tempuh sperma untuk
mencapai fertilisasi tuba
fallopi pendek
3. Lebih banyak oosit berada di
tuba fallopi meningkatkan
fertilisasi
4. Teknik sederhana dan murah
5. Lebih diterima oleh kelompok
umat beragama

Kekurangan

Angka keberhasilan < IVF

Jika siklus gagal sedikit
informasi yg di dapatkan,
daripada IVF

Prosedur Pelaksanaan Intrauterine Insemination (IUI)

1. Siklus Natural /
Tanpa Stimulasi
(Natural Cycle)

2. Siklus
Stimulasi
(Stimulated
Cycle)

Usia

muda

Infertilitas

akibat
faktor sperma

Jumlah Oosit
produksi steroid untuk
terjadinya fertilisasi dan
implantasi

Siklus Stimulasi
(Stimulated Cycle)
1. Klomifen Sitrat

Siklus Stimulasi
(Stimulated Cycle)

2. Injeksi FSH
(Follicle
Stimulating
Hormone)

Dosis awal FSH : 75-100

IU
Bertambahnya usia >
40 th dosis awal
dinaikkan 225 300
IU

Siklus Stimulasi
(Stimulated Cycle)
Mengaktifkan GnRH di
Hipotalamus menstimuli
hormon gonadotropin
Pertumbuhan dan
perkembangan folikel
Dosis awal 50 -100 mg, pd hari ke2. (5 hari)
Hari ke-8 USG, sampai folikel 1718 mm injeksi HCG 36 jam
IUI dilakukan

3. Kombinasi
Klomifen Sitrat &
Injeksi FSH

Angka Keberhasilan IUI pada Wanita

Campana et al, melaporkan angka keberhasilan hasil IUI adalah
18,7% per pasien dan 5,6 % per siklus.
Frederick et al, angka keberhasilannya adalah sekitar 21 % per
pasien dan 10 % per siklus.

Menurut Studi
Reindollar et al,

1. Menjalani 3 siklus IUI, Klomifen sitrat,

gonadotropin, FIV (fertilisasi In Vitro) angka
keberhasilan 7,6 %, 9,8% dan 30,7 %
2. Menjalani FIV setelah IUI dengan Klomifen sitrat
(rasio hazard 1.25, IK 95% 1.00-1,56).

Sperm Preparating for Intrauterine

Insemination
Artificial
Insemination by
Husband (AIH)
A. Semen Collection

Donor Insemination
(DI)

B. Sperm
Preparating
Technique

1.
2.
3.
4.

The Sperm Count

Sperm Washing
Swim-up Method
Density Gradient
Centrifugation

A. Semen Collection
1. Artificial Insemination by Husband (AIH)
. The male partner will be required to produce a sample
of semen by ejaculating into a sterile container.
. Two to three days abstinence from intercourse /
masturbation is preferred prior to the sample collection
day.
. Clinics often provide a room so that this sample may
be produced in private, but some men prefer to collect
the semen at home and deliver it to the clinic.
. As being used that day it must not be frozen or
refrigerated, and it needs to arrive promptly at the
clinic within a couple of hours.

Gambar 2.3 : sterile

container.

A. Semen Collection
2. Donor Insemination (DI)
IUI can also be done using donor sperm, either from an anonymous
or known sperm donor (knows as DI or donor insemination.
Insemination with donor sperm is used when there is no male partner
or the male partner does not produce sperm, when the sperm is of
very poor quality or if there is a high risk of passing on genetic
disease.
Sperm are usually frozen ahead of time and screened for sexually
transmitted diseases (e.g HIV/AIDS/Hep/HepC) and any genetic
disorders

Dalam pelaksanaanya, TRB (Teknik Reproduksi Berbantu) di

Indonesia harus merujuk pada Panduan Etika dan Profesi
Obstetri dan Ginekologi
Pasal 27
Donasi materi genetik baik berupa gamet (sperma, oosit) maupun
zigot/praembrio tidak dibenarkan
Penjelasan
Yang dimaksud materi genetik (genetik material) ialah gamet (sperma,
oosit) dan zigot atau praemrio yang siap ditransfer. Donasi materi genetic
ini sering dipakai sebagai salah satu teknik untuk mengatasi infertilitas,
meskipun dapat menimbulkan masalah sosial, religious, dan etis.
Donasi materi genetik tersebut akan memutuskan garis silsilah keluarga
dan dianggap analog dari zina. Oleh karena itu teknik inseminasi dari
donor (AID) dan ibu titip (surrogate mother) tidak dapat dibenarkan.

1.
2.
3.
4.

B. Sperm
Preparating
Technique

The Sperm Count

Sperm Washing
Swim-up Method
Density Gradient
Centrifugation

WHY SPERM
PROCESSING?

To remove the seminal plasma, dead sperms, debris, round

cells

To concentrate the more active sperms

To improve their motility

Role of free oxygen radicals ROS (Reactive Oxygen Species)

DISPOSA
BLES
Semen collection
containers
14ml conical/6ml
round bottom tubes
Pasteur pippette
IUI Cannulla
BD Syringe
Cryovials
Powder free gloves
70% Ethyl Alcohol for
cleaning the Lab

TYPES OF MEDIA

Single Layer Density

gradient
Double layer Density
gradient
Wash media / HAM
F10 / EBSS
Cryo preservation
media

B. Sperm Preparating
Technique

1. The Sperm
Count

Parameter
Semen Volume (ml)

Lower reference
limit
1,5 5,0

Seminal PH

>7,2

Before and after the

treatment of the seminal fluid,
the following parameters
must be evaluated in line with
the WHO manual 2010.

Viscosity

<3 (scale 0-4)

Sperm concentration

20 million/ml

Forward progression

<2 (Scale 0-4)

Analisis sperma ulang u/

mengkonfirmasi
pemeriksaan sperma
abnormal.

Sperm morphology
(Normal forms, %)

>50% normal 21
>30% normal 22
>14% normal 9

<5 million / ml

3 bulan pasca pemeriksaan

sebelumnya sehingga
proses siklus pembentukan
spermatozoa

Total Sperm number (per

ejaculate)
Percent motility

Round cell

Sperm Agglutination

20 million/
ejaculate
>50%

< 2 (scale 0-3)

Tabel 2.3. Analisa Semen : Standar minimal untuk semen normal

berdasarkan kriteria WHO (2010).

1. The sperm count

Pemeriksaan Computer-Aided
Sperm Analysis (CASA)
Untuk melihat jumlah, motilitas,
dan morfologi sperma,
pemeriksaan ini tidak dianjurkan
karena tidak memberikan hasil
yang lebih baik dibandingkan
pemeriksaaan secara manual.

CASA
System

1. The sperm count

Pemeriksaan fungsi endokrinologi
pasien dengan konsentrasi sperma < 10 juta/ml,
Bila secara klinik ditemukan bahwa pasien menderita
kelainan endokrinologi.
Pada kelainan ini sebaikknya dilakukan pemeriksaan
hormone testoteron dan FSH serum.

2. Sperm Washing
The simple wash technique is usually used when the semen
sample has optimal parameters.
This technique is often used to prepare sperm cells for
intrauterine insemination because it produces very high yields of
spermatozoa.

2. Sperm Washing
washing of the semen with sterile medium added with
human albumin.
the entire volume is divided in fractions of not more than 2 ml
into cenftrifuge tubes.
gently mix with sterile pipette. After that, the sample are
sentrifuged at 300g for 10 minutes
The pellet is resuspended in 1 ml of the medium, gently mixed
and centrifuged again for 5 min at 300 g, the supernatant is
removed again and the final pellet is resuspended in steril
medium.

2. Sperm Washing

VIDEO
Sperm Washing

3. Swim-up Method
The swim up is the most common technique for sperm
preparation.
This method is used if the semen sample is very poor
By this technique, the sperm are selected on their
motility and the capability to swim out of the seminal
plasma.

3. Swim-up Method

Semen is diluted with 1:2 ratio

of
culture
medium
and
centrifuged at 250-300g for 5-7
min.
The supernatant is removed
leaving the pellet.
Pipette 0.8-1 ml of media into a
new test tube.
Incubated at 37o for 45-60 min.
Placement of tube at 45o angle
sperms to swim-up of the sperm
and to reach the medium
After that, tubes must be
returned vertical position and 1
ml of supernatant of each tube
removed
Aspirating sperm form the upper
meniscus downwards with sterile
pipette.

VIDEO
Swim-up Method

3. Swim-up Disadvantages
Centrifugation, has been shown to generate ROS (Reactive
Oxygen Species)
The amount of motile spermatozoa low only 5-10% of the sperm
cells
When a concentrated cell pellet is used, some motile
spermatozoa may be trapped in the middle of the pellet and thus
may not travel as far as sperm cells at the edges of the pellet.

4. Density Gradient Centrifugation

This is the preferred technique to select the greater

number of motile spermatozoa in cases of severe
oligozoospermia, teratozoospermia.
In this method, good quality sperm can be separated
form dead sperm, leukocytes and the other components
of the seminal plasma

4. Density Gradient Centrifugation

Upper phase 4 ml of density gradient

medium to 6 ml isotonic sterile medium
(BWW, Earle, Ham f-10 or HTF)
supplemented with HAS (Human Serum
Albumin)

the lower phase (80%) is prepared by

adding 8 ml of density gradient medium to
2 ml of isotonic sterile medium.

Volume of the sample not more than 1

ml of semen pertube.
1 ml semen the upper layer
centrifuged at 300 g for 15 -20 minutes.

The concretration and the motality

after preparation can be determined.

VIDEO
Density Gradient Centrifugation

4. Density Gradient Centrifugation

Advantages of density gradient
centrifugation
Density gradient centrifugation requires
maximally a thirty-minute centrifugation.
It takes less time than the swim-up
technique which requires one-hour
incubation.
Density gradient centrifugation is
relatively easy to perform under sterile
conditions.
Spermatozoa from oligozoospermic semen
can be effec- tively separated with density
gradient centrifugation.

Disadvantages of density gradient

centrifugation
Production of good interphases between
layers can take some time.

Density gradient centrifugation eliminates

the majority of leukocytes in the ejaculate.

There is a risk of contamination with

endotoxins.
Some scientists have claimed that density
gradient centrifugation negatively affects
sperm DNA integrity. For instance.

Resiko Inseminasi
Intrauterin
Penelitian laporan 38 seri IUI 5 dari 3129 memiliki
komplikasi infeksi
Infeksi pada wanita yang menjalani IUI 1,83 per 1000
Resiko hamil ganda
Penelitian Cochrane : IUI untuk Laki-laki subfertil data
yg dimiliki tidak cukup secara statistik u/ mengevaluasi
akhir seperti; keguguran, hiperstimulasi ovarium
sindrom & KET.

TERIMAKASIH