Till few years back options for the treatment of fertility were
limited but, with the advances in assisted reproductive techniques (ART) the
couples who were termed as sterile were able to procreate. ART consists of
various methods that are used to manipulate oocytes and sperm to overcome
infertility. The first successful artificial insemination was done in early
1770’s and the first In vitro fertilization (IVF) was done by Steptoe and
Edwards in 1983. In 1992 intra cell sperm injection (ICSI) of a single sperm
into an oocyte was performed by Palermo et al. IVF is said to be
the backbone of ART and in the last 20 years lot of advances have been made in
the field of assisted reproductive technology (ART) viz.
Superovulation:
The hormonal
intervention by which the ovulation is increased during ovulatory cycle due to
which multiple oocytes are released is termed as superovulation. For the
stimulation of ovaries; exogenous human gonadotropins are used which have FSH
and LH activity e.g. human menopausal gonadotropin (HMP). Recombinant FSH and
LH are also used for ovarian stimulation these days. Ovarian stimulation with
gonadotropins is combined with Gonadotropin releasing hormone (GnRH) agonist
e.g. leuprolide acetate so that the endogenous LH is suppressed and premature
leutinization of follicle is avoided.
Oocyte retrieval:
Oocytes are
retrieved transvaginally under the guidance of ultrasound after sedating the
patient 34-36 hours of hCG administration. This timing is important for the
maturation ofoocyte and also to minimize the spontaneous ovulation. After
retrieval, oocytes are separated from the follicular fluid and are separated
according to morphology and mature oocyte which is characterized by the
presence of first polar body is separated as these are best for fertilization
in vitro.
In vitro
maturation (IVM):
In this process, the immature oocytes are retrieved from the
unstimulated ovaries by aspirating them from antral follicles during the early
phase of menstrual cycle under the guidance of an ultrasound. After retrieving,
eggs are matured in the laboratory for 24-48 hours. After maturation these eggs
can be used for fertilization using ICSI etc. IVM is very helpful in patients
having polycystic ovarian syndrome (PCOS) and for preserving fertility in
cancer.
Embryo transfer (ET):
For embryo-transfer a sample of sperm is prepared on the same day of
oocyte retrieval. This sperm sample is processed by percoll gradient
centrifugation technique and is incubated in protein rich medium for 4 hours to
initiate capacitation. Now, the mature oocytes are inseminated and are examined
after 18 hours to check fertilization which is confirmed by the presence of 2
pronuclei and 2 polar bodies in the perivitelline space. After 72 hours when
embryo reaches 4-8 cell stage; they are transferred into the uterus.
Microsurgical epididymal sperm aspiration (MESA):
It is an epididymal sperm retrieval technique in which each epididymal
tubules are identified under operating microscope and sperms are aspirated or
micro punctures are made for obtaining a fine amount of sperm. Alternatively,
tubules can be incised and fluid can be gathered. As sperms in the epididymal
fluid are highly concentrated therefore, sperms are retrieved in microliters
only. The retrieved sperms can be used immediately or cryopreserved.
Percutaneous epididymal sperm aspiration (PESA):
In PESA, epididymal sperms are aspirated without surgical opening of
scrotum. The patient is given general anaesthesia (GA) and a butterfly needle
attached to a 20ml syringe is inserted into the caput epididymis assuming that
sperm must be there and sperms are aspirated and the needle is withdrawn
gently. The aspiration is done repeatedly till surgeon gets sure of the amount
of epididymal fluid.
Testicular sperm aspiration (TESA):
In this technique, patient is given GA and a small incision is made in
the scrotal skin and with the help of spring loaded needle sperms are collected
from the testes.
Testicular sperm extraction (TESE):
In this process, under GA a small portion
of tissue from the testes is removed and viable sperm cells are extracted from
that tissue. The amount of sperm obtained is low and any injury to testes can
lead to testicular infarction.
Testicular sperms can also be retrieved during Vasovasostomy
and Vasoepididymostomy if motile sperms are found during the operative
procedure.
Intra cytoplasmic sperm injection (ICSI):
It is an invasive microinjection technique in which single sperm is
directly injected into the cytoplasm
of a mature oocyte. During ICSI, the microinjection pipette containing the
single sperm pierces the zonapellucida at 3 o’clock position and is pushed deep
into the cytoplasm; sperm is injected and needle is removed. After 16 hours of
injection, oocyte is examined for fertilization and if successful, embryo is
transferred after 1-3 days of fertilization. ICSI has replaced largely subzonal
sperm injection (SUZI), partial zona dissection (PZD) and zona drilling (ZD).
Pre-implantation genetic diagnosis (PGD):
PGD allows identification of specific genetic disorders before embryo
transfer. For PGD, the embryo at 8-cell stage is used for biopsy and during biopsy
the zonapellucida is pierced and a single blastomere is removed for analysis.
Along with blastomere biopsy, polar body and trophectoderm biopsy can also be
done. For chromosome analysis fluorescence in situ hybridization (FISH) and
polymerase chain reaction (PCR) are used and for identification of whole
chromosome abnormalities; comparative genomic hybridization (CGH) is used.
Assisted hatching:
Inability of blastocysts to hatch from zonapellucida is one of the
main factors responsible for implantation failure of IVF. In this technique,
the embryo to be transferred is held using a micropipette and with the help of
acidic solution or laser or other mechanical methods; a small hole is created
in the zonapellucida. The embryo is then washed to get rid of the medium and is
placed in incubator until ET.
Cryopreservation:
In this technique, cells or tissues are preserved at sub-zero
temperature. Now days, apart from embryo and sperm preservation; oocytes and
ovarian tissues can also be cryopreserved in women with risk of premature
ovarian failure e.g. in women with Turner’s syndrome. For cryopreservation,
ovarian cortex is isolated and cut into strips of 10mmx5mmx1mm dimensions and
are then incubated with cryoprotectants e.g. dimethyl sulfoxide (DMSO) and are
frozen using programmable freezer.
Vertification:
It is an alternative method of preservation using the benefits of
cryopreservation. Cryopreservation leads to ice formation but, in
vertificationcryoprotectants are added prior to cooling and thus by rapid
cooling ice formation is avoided and cells are stored in a glass state. Process
of vertification is still experimental.
Dr.Bharati Sood
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