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  Section: General Biotechnology / Animal Biotechnology
 
 
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Manipulation of Reproduction and Transgenic Animals

 
     
 

In Vitro Fertilization and Embryo Transfer in Humans
Due to several complicacies associated with humans, there has been the birth of in vitro fertilization technology. The IVF technology is a boon to childless couples. Initially this technique was pioneered by Prof. Robert Winston during 1970s which was later applied by P. Steptoe and R. Edwards for the production of first test tube baby, Louse Joy Brown, who was born on July 25, 1978. Since then more than 25,000 test tube babies have been produced so far through out the world.

Infertilities in Humans
There is a large number of reasons for developing infertilities in humans. By using IVF many more infertilities have been treated.

Male sterility
In a fertile human the total count of sperms should be 15-20 million per ml. When the number of sperms decreases, the person is called oligospermic and the condition as oligospermia. Similarly, when there is very low number of motile sperms, the patient is called azoospermic and the phenomenon as azoospermia. Generally in azoospermic humans the number of non-motile sperms is high. In addition, for IVF, the presence of motile sperms should be around 10-20,000 per ml.

Female sterility
There are several reasons and types of female infertility; few of them are : (i) non-functional (inaccessible or absent) ovaries (some times ovaries of female are non-functional or inaccessible, therefore, healthy oocytes will not be produced. In such cases oocytes can be procured from donor for IVF), (ii) non-functional (or absent) uterus (in this condition the oocytes are obtained from female and subjected to IVF, and embryo transferred to surrogate mother for pregnancy and further development), (iii) idiopathic infertility (no definite cause is known for idiopathic infertility. Possibly it may be due to abnormal fertilization or failure in fertilization. Therefore, IVF followed by embryo transfer will cure this infertility), and (iv) tubal infertility (fallopian tubes receive oocytes from follicles of ovaries for fertilization. Any deformity in fallopian tubes results in tubal infertility. IVF followed by embryo transfer in uterus has replaced the function of fallopian tubes. Therefore, the patients will not be subjected to surgical treatment).

Who Benefits IVF
It is not likely that any patient of any age or any group of the society can go for treatment for IVF. There are several prerequisite conditions applicable for male and female partners. The male should not be azoospermic; however, if he is azoospermic and desires for a baby, he can procure semen either from sperm bank or elsewhere. Besides, the female partner should also be healthy and fit for IVF because it is she who is to bear the pregnancy. Therefore, the female must be such that (i) any surgical procedure should not create any trouble to her, (ii) her ovaries should be accessible for oocytes, (iii) her uterus should accept and bear pregnancy for the required period of about nine months, and (iv) her cervical canal should be easily negotiable for embryo transfer.

How the Patients for IVF Treated
First of all the female aspirant for IVF is suggested to keep the records of her menstrual cycle upto six months. This information supports the doctors to take fruitful decision for the possibility of IVF. The female patient is hospitalized to ensure the date for collection of urine for the presence of luteinizing hormone (LH) surge, and to fix date for administrating human choripnic gonadotrophin (hCG). The hCG controls the final stage of formation of follicle and oocytes. The oocytes may be recovered during any menstrual cycles i.e. natural cycle, stimulated cycle or controlled cycle. According to the nature of three cycles, the treatment for IVF is recommended.

 

Content

Manipulation of reproduction in animals

 

Artificial insemination

 

 

Semen collection and its storage

 

 

Ovulation control

 

 

Sperm sexing

 

Embryo transfer

 

 

Multiple ovulation (superovulation)   

 

 

Multiple ovulation with embryo transfer

 

 

Embryo splitting

 

 

Embryo sexing

 

In vitro fertilization (IVF) technology

 

 

In vitro Maturation (IVM) of oocytes

 

 

Culture of in vitro fertilized embryos

 

Embryo Cloning

 

 

Quadriparental hybrid

 

 

Nuclear transplantation (Dolly)

 

 

Embryonic stem cells

In vitro fertilization and embryo transfer in humans

 

Infertilities in humans

 

 

Male sterility

 

 

Female sterility

 

Who benefits IVF

 

How the patients for IVF treated

 

 

Indicators of ovary stimulation

 

 

Oocyte recovery and uptake

 

 

Semen preparation

 

IVF and embryo transfer

Transgenic animals

 

Strategies for gene transfer

 

 

Transfer of animal cells/embryo

 

 

Treatment through microinjection

 

 

Targeted gene transfer

 

Transgenic animals

 

Transgenic sheep

 

Transgenic fish

 

Animal bioreactor and molecular farming

Application of molecular genetics

 

Selected traits and their breeding into livestock

 

 

Diagnosis, elimination and breeding strategies of genetic diseases

 

Application of molecular genetics in improvement of livestock

 

 

Hybridization based markers

 

 

PCR-based markers

 

 

Properties of molecu­lar markers

 

 

Application of molecular markers

 

 

Transgenic breeding strategies


During natural cycle, spontaneous LH surge is found out at 3-6 h intervals by taking urine or plasma sample. Under the influence of LH surge fully developed follicles of both the ovaries burst and ova collected in fallopian tubes. In natural cycle, only one ova at a time is formed. Secondly, the stimulated cycle is created by administrating clomiphene and/or human menopausal gonadotrophin (hMG). Clomiphene (150 mg) per day is given for the 5th to 9th day of the cycle. However, the duration may be prolonged for 10 minutes but there will be more risk of uterus receptivity and abortion. The hMG alone or along with Clomiphene is also given that does not allow the high rate of abortion. But several abnormalities are also caused by hMG. Consequently multiple follicles and oocytes, are stimulated instead of one. At this time hCG is also given to female patients so that inhibition of LH surge can be checked. The disadvantages associated with these are the ultrasonographic assessment of the hormonal effect and development of some of abnormal oocytes that result in abnormal pregnancy. Thirdly, hCG is administered at the optimum stage of maturation that arrests the development of follicle. However, it is difficult to predict the optimum time for hCG administration that varies for different preovulatory follicles. Patients need not stay in hospital for a long duration because the doctor can (to his own and patient*s convenience) recover oocytes through laparoscopy. His success rate in oocyte recovery, embryo transfer and pregnancies are achieved by the stimulated and controlled cycles.

Indicators for ovary stimulation
It is very necessary to monitor the stimulation of ovary so that the oocytes can be collected at right time of development. There are several parameters which are used as indicators for stimulation of ovary, these include: (i) prediction of LH surge on the basis of menstrual cycle, (ii) rise in temperature of body during preovulatory and postovulatory days, (iii) changes in cervical mucus as it is secreted five days before LH surge, goes maximum a day before LH surge and falls sharply thereafter, (iv) estimation of progesterone, (v) levels of oestrogen in urine, etc.

Oocyte recovery and culture
Oocytes are recovered through recently developed equipment called laparoscopy. Using microscopic equipment the follicles can be observed and aspirated with the aspirating apparatus which consists of 23 cm long needle made up of stainless steel (with internal diameter of 1.6 mm and external diameter 2.1 mm) and 52 cm long tubing. The total space between needle and tubing is 1.2 ml. The ovulating follicles are selected for aspiration. It consists of ova of less than 1.5 cm. From such follicles fluid (4-12 ml) is aspirated by using needle and tubing. After aspiration wall of follicle gets collapsed. Fluid is of straw colored some times it contains blood also. When there appears blood, heparinised culture medium should be added in tube so that blood clotting may be prevented. However, if fluid contains blood, ova could not be easily identified and isolated.

The aspirate is observed under the microscope for the presence of oocytes. If ova are not present in aspirate, the heparinised culture medium should be reflushed into follicles to get oocytes. The oocytes are identified under the microscope and incubated for 5-10 h depending on maturity of oocytes and follicles. The media used for oocyte culture and IVF are modified Ham's F10 medium, modified Whitten's medium, Earl's solution and Whittingham's T6 medium. Any one of these media may be used.

Semen preparation
The semen is procured either (from husband of female partner or from semen bank or elsewhere. Husband's semen is collected at site when required through masturbation 60-90 minutes prior to insemination. Semen is subjected to centrifugation; sperm pellets are recovered and transferred to culture medium for suspending it again. It is then centrifuged and semen pellets are retransferred to culture medium. The sperms are incubated at 37°C for 30-60 minutes. The most active spermatozoa float at surface. Therefore, samples for IVF should be taken from surface of culture medium containing spermatozoa.


In vitro Fertilization and Embryo Transfer
Insemination is done by mixing about 1 ml culture medium containing oocytes with 1 ml of semen preparation (that contains 10,000-50,000 spermatozoa/ml). Oocytes are incubated for 12-13 h. Thereafter, they are observed keeping them in a Petri dish for the presence of two pronuclei and two polar bodies. Besides, the abnormal fertilized oocyte contains more than two pronuclei and granulation in cytoplasm. First cleavage occurs after 24-30 h of insemination. Delay in cleavage after insemination confirms that the embryo is abnormal. Thus, the abnormal embryo is discarded; only normal embryo is used.

After fertilization, embryo should not be detained for el long time in vitro. It should be transferred to treated recipient female when it is in 2-4 cell stage. However, success has been achieved with transfer of 1-16 celled embryos. However, early embryo fails to survive in uterus. This stage is very critical, therefore, much care should be taken.

  Outline of embryo transfer in treated female human.
 

Fig. 7.6. Outline of embryo transfer in treated female human.

 
Some times, multiple embryo transfer is also done. It may increase the chance of embryo survival, but results in multiple pregnancy to which the female does not like. The transferred embryo stays in fallopian tube up to 8-16 cell stage, thereafter moves to uterus. Embryo of prolonged stage in vitro should not be transferred because of less chance of survival and pregnancy.

Embryo transfer should be done in an operation theatre. No anaesthesia should be given to female. She is given only Diazepam (Valium) of 10 mg orally before embryo transfer. The embryo is to be transferred through cervical canal; therefore, the patient is placed in lithotomy (i.e. in the position of knee and chest with tilting the head down) so that the fundus should be in lower position than cervical canal (Fig. 7.6). The cervix is visualized by inserting a sterile bivalve vaginal speculum. Then uterine, cavity is aligned to cervical canal by manipulating the uterus. Distance between fundus and external cervical is earlier measured through ultrasonography. The embryo is drawn into a teflon catheter along with 10 ml of tissue culture medium. The catheter is gently inserted into culture medium. It should be made sure that the embryo has been transferred or not. The catheter is observed under microscope. Absence of embryo in catheter ensures for its transfer in uterus. Wrong placement of catheter or high amount of medium will result in tubal pregnancy or expulsion of embryo from uterine cavity. Therefore, care should be taken for proper handling. After embryo transfer, the female is allowed to take rest for about 2-7 h and abstain from intercourse for about 10 days. The patient follows the advice of doctor and undergoes for regular check up.


 
     
 
 
     



     
 
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