In Vitro Fertilization (IVF)

In vitro fertilization (IVF) is used clinically in cattle and humans. In cattle, oocytes are
recovered from cows in vivo up to twice weekly by transvaginal ultrasound-guided follicular
puncture.

In vitro fertilization (IVF) is an assisted reproductive technology (ART) initially introduced

by Patrick Steptoe and Robert Edwards in the 1970s to treat female infertility caused by
damaged or blocked fallopian tubes. This major breakthrough in embryo research has
provided large numbers of women the possibility of becoming pregnant, and subsequent
advances have dramatically increased their chances. IVF is a laboratory procedure in which
sperm and egg are fertilized outside the body; the term “in vitro” is Latin for “in glass.”
Although the procedure was not successfully established until the last quarter of the twentieth
century, the history behind the development of IVF dates back much further. The first attempt
at IVF on mammalian eggs was performed by Viennese embryologist Samuel Leopold
Schenk in 1878. Working with rabbit and guinea pig ova, Schenk noted that cell division
occurred in cultures after sperm were added to ova. In 1934 Gregory Pincus and Ernst
Vinzenz Enzmann also attempted in vitro fertilization with rabbits. They claimed to have
produced the first successful pregnancy using IVF, but later analysis of their study suggests
that their fertilization technically occurred in vivo, or “in the body,” rather than in vitro, “in
glass.” They implanted the eggs into the rabbit’s uterus after only twelve hours, before the
eggs had fully matured, and fertilization actually occurred inside the body. The next
milestone was in 1951, when two scientists working independently, Colin Russell Austin in
Australia and Min Chueh Chang in the United Sates, demonstrated that spermatozoa need to
mature through certain stages before they develop the capacity to fertilize. By 1959 Chang
was able to successfully use IVF to impregnate a rabbit. Significant progress in developing a
successful IVF technique with human embryos, however, would have to wait until the 1970s.
Patrick Steptoe, a practicing gynecologist at Oldam General Hospital who pioneered the use
of laparoscopy in gynecology, teamed up with Edwards, a professor of human reproduction at
Cambridge University, to try to achieve a successful pregnancy in humans using IVF. Their
collaboration started in 1968 when Edwards attended a lecture Steptoe gave on laparoscopy
at the Royal Society of Medicine in London. They initially achieved successful fertilization
and cell division of eggs in vitro (in a petri dish) with freshly extracted semen, but were
unable to successfully implant the fertilized egg into the female uterus until 1978. They
manipulated hormone levels in the female until the eggs were fully mature and then extracted
several eggs from the ovaries through laparoscopy, an invasive technique requiring entry
through the navel. The doctors fertilized the eggs in vitro, and waited until the fertilized eggs
divided into eight cells before implanting them into the female uterus (up until the mid-
1970s, they had waited until the fertilized egg divided into 100 cells before implantation). In
1976 Edwards and Steptoe began working with an infertile couple, Lesley and John Brown.
In the successful attempt, Edward and Steptoe transferred a fertilized egg at midnight, the
time at which the egg was mature—accidental timing that they later discovered was critical
when they realized that diurnal cycles of hormonal levels are crucial to the success of the egg
implanting in the wall of the uterus. On 25 July 1978, Leslie gave birth to Louise Brown, the
first “test tube baby.” Since the birth of Louise Brown, over three million babies have been
born as a result of IVF and other assisted reproductive technologies, and the technique has
improved as well. Laparoscopy is no longer used to extract eggs from the ovaries. Instead,
physicians use transvaginal oocyte retrieval; that is, with a sonogram to visualize what they
are doing, they guide a needle through the vaginal wall, and enter the ovaries to extract the
eggs. By using this method, the risks associated with the anesthesia required for laparoscopy
as well as the costs of the procedure are considerably reduced. Physicians now begin giving
women hormone therapy two weeks prior to retrieving the eggs to increase the chance of
recovering several healthy, mature eggs. Hormone therapies are usually administered through
oral medications such as clomiphene citrate, also known as Clomid. Ultrasounds and blood
tests are used to determine the optimal time for egg retrieval (when the eggs are almost ready
for fertilization). Once physicians obtain the eggs, they then place them in a petri dish with
sperm for fertilization. Usually about seven to nine eggs are fertilized. If a male fertility
problem exists such as sperm immotility or a low sperm count, intracytoplasmic sperm
injection (ICSI) is commonly used to combat the problem. With ICSI, the physician manually
injects a sperm into an egg with a needle to fertilize it. The number of fertilized eggs
physicians place in the uterus in hopes that at least one will implant varies with the physician
conducting the procedure. Physicians consider different factors with each couple, such as the
number of trials the couple can afford to attempt. The remaining embryos are preserved
through cryopreservation, frozen for future use in case the first few embryos fail to implant in
the uterus wall on the initial attempt. Once the fertilized eggs each divide into eight cells,
they are placed in a catheter and inserted through the cervix into the uterus (this usually
occurs from two to three days after retrieving the eggs and follows the same timeline as if the
eggs were fertilized naturally). When Edwards and Steptoe successfully helped the Browns
give birth to Louise, the first “test tube baby,” many ethical debates arose. Many of these
issues still surround embryo research today, and they include who essentially “owns” the
embryos as well as whether scientists should be allowed to perform experiments for stem cell
research with the extra cryopreserved embryos that are not implanted. Many religious groups,
the Roman Catholic Church being the most prominent, are strongly against IVF because it is
not considered natural conception. IVF has greatly advanced embryo research as well as
helped many infertile couples conceive when adoption was once their only option for
children. Though there are other similar assisted reproductive technologies (ART) such as
gamete intrafallopian transfer (GIFT) and zygote intrafallopian transfer (ZIFT), IVF is
currently the most popular. The development of intracytoplasmic sperm injection (ICSI) has
improved success rates.

Summary

Many factors influence the final oocyte maturation, fertilisation, and early embryo
development, and there are both similarities and differences between species. When
comparing the advancement of assisted reproductive technologies (ARTs), the development
in the bovine species is not far behind the medical front, with around one million in vitro-
produced bovine embryos each year. This rate of progress is not seen in the other domestic
species. This study aims to give an overview of the development and specific difficulties of in
vitro embryo production in various domestic animal species, with the main focus on cows,
pigs, and cats. In production animals, the aim of ARTs is commonly to increase the genetic
progress, not to treat reproductive failure. The ARTs are also used for preservation of genetic
diversity for the future. However, specifically for oocyte maturation, fertilisation, and early
embryonic development, domestic mammals such as the cow and pig can be used as models
for humans. This is particularly attractive from an animal welfare point of view since bovine
and porcine oocytes are available in large numbers from discarded slaughterhouse material,
thereby decreasing the need for research animals. Both for researchers on the animal and
human medical fronts, we aim for the development of in vitro production systems that will
produce embryos and offspring that are no different from those conceived and developed in
vivo. Species-comparative research and development can provide us with crucial knowledge
to achieve this aim and hopefully help us avoid unnecessary problems in the future.
Introduction

It is easy to assume that in the beginning of our embryonic development we animals are all
very much alike. In some aspects this is true, but there are major differences between species,
and even between mammals. In many ways, cows and humans are similar in the advancement
of assisted reproductive technologies (ARTs) and in vitro fertilisation (IVF), but many
species still lag far behind. This review attempts to give an overview of the development and
specific difficulties of in vitro embryo production, including IVF, in various domestic animal
species, with the main focus on cows (bovines), pigs (porcines), and cats (felines).
Commonly in production animals, the focus is not to overcome reproductive failure but to
enhance the genetic progress, both by selection of the most genetically valuable individuals
and by increased number of offspring from these individuals. With species-comparative
knowledge, our possibilities to understand the mechanisms behind various aspects of the final
oocyte maturation, fertilisation, and early embryonic development in vitro will increase. IVF-
associated ARTs such as artificial insemination, which is used successfully and to a very
large extent in developed countries, sperm capacitation, intracytoplasmic sperm injection
(ICSI), embryo transfer (ET), and cloning fall outside the scope of this review and are only
briefly mentioned.

An overview of procedures
The procedures of in vitro oocyte maturation (IVM), in vitro fertilisation (IVF), and the
first days of in vitro embryo culture (IVC) are collectively termed in vitro embryo
production (IVP). In common domestic animals we usually retrieve immature oocytes
from the ovaries, either after normal castration where ovaries are removed, in ovum
pick-up (OPU) procedures in live animals, or after the death of the animal (due to
slaughter, euthanasia, or after fatal trauma). In experimental settings or within some
commercially IVP companies, in vivo maturation procedures may occur. Since animal
ovaries are not considered fit for human consumption in most countries, they are a
waste product in the meat industry and can easily be retrieved fresh in large quantities
from consenting abattoirs. Routine castrations or neutering/spaying of pets are
performed on the explicit wishes of the owners and according to recommendations of
veterinarians due to various reasons that can include prevention of unwanted offspring
as well as changes in temperament or behaviour connected to hormonal actions in an
intact animal. Normal routine castrations are performed daily in most veterinary
clinics, and also here the ovaries are discarded as waste and can be collected for IVP
purposes. This relatively easy access to large quantities of ovaries without having to use
research animals makes species like the cow and pig perfect candidates for IVP
research, spanning from testing of in vitro media and methodological improvements to
controlled trials of newly emerging risk substances like environmental pollutants found
in humans and our environment. However, with the use of these discarded ovaries there
are no possibilities to control cyclicity or follicular growth that would be possible in
research animals. Due to the presence of continuous follicular waves in most domestic
animals , the ovaries will always contain cumulus oocyte complexes (COCs). Therefore,
the approach is usually to retrieve relatively immature COCs and perform the final
maturation in vitro. The selection process starts with aspiration of follicles of a certain
size range depending on species, commonly by hand with needles and syringes. The
follicles aspirated should not be too small, since they would contain COCs that are too
immature and difficult to mature to MII stage . On the other hand, the follicles should
not be too large since they could contain COCs that are too mature, with already-
expanded cumulus cells or atretic oocytes, that would make them unsuitable to include
in the process (Figure 1). After the aspiration or OPU all COCs are collected, and only
COCs of good quality are selected for the IVM process (Figure 1). The ideal bovine
COC is light and transparent, and has a compact multilayered cumulus investment and
a homogeneous ooplasm . In both pigs and cats the COCs are darker than in the bovine,
but otherwise the same criteria are used. During IVM, media are supplemented with
LH and FSH, and in some cases oestradiol, growth hormones, and insulin. After IVM
the medium is changed to promote sperm capacitation and fertilisation. The duration of
fertilisation ranges between a few hours and up to one day, depending on species, and is
followed by IVC where the medium is promoting the early embryo development.
Embryos are commonly evaluated and classified according to the International Embryo
Technology Society (IETS) guidelines. A common time point for transfer back to a
recipient animal is in the stages of morula or early blastocyst, but later blastocyst stages
as well as earlier embryos could also be transferred (Figure 2). In order to avoid the
risk of transferring diseases, embryo transfer teams (mainly working with bovines)
must also follow the recommendations of the IETS.

culture took place in oviducts from rabbit or sheep and later on after co-culture with
oviductal cells. Gradually, supporting cells were excluded as formulas for the media
improved. Chemically defined media have given valuable insights to our understanding
of the impact of various substances on oocyte maturation, fertilisation, and early
embryo development and the possibility to avoid unknown macromolecules. Moreover,
potential contaminants from e.g., serum in culture conditions are of great importance.
ARTs have over the years developed from invasive surgical procedures to ultrasound-
guided techniques in many species. IVM is still a major hurdle to overcome in most
species, and another difficulty has been the capacitation process. Different species, as
well as male individuals within the same species, have differences in substances and
concentrations of these that can induce capacitation. Heparin, serum albumin,
epinephrine, penicillamine, hypotaurine, coffein, bicarbonate, and calcium are some of
the substances that can induce capacitation. It is quite obvious that there is a large step
from maturing and fertilising oocytes in vitro to actually being able to produce live
offspring after both procedures since this seems to take over 20 years in most species. It
is worthy of note that, even though cases of successful IVF with live offspring have been
reported in dog and horse, such reports have been few and have proven difficult to
repeat.

IVP in cattle

Today the cattle IVP industry is expanding at a high rate and does not show any signs of slowing down. According to
the International Embryo Technology Society (IETS) almost one million transferable in vitro-produced cattle embryos
were produced all over the world in 2017 (Table 2). Unfortunately, not all countries contribute to this report, and these
numbers are therefore under-estimated. There has been a remarkable increase since 2016 when only about half a
million bovine embryos were produced. Most of the transfers were carried out in Brazil, closely followed by the USA.
In Europe, there were some 50,000 transfers of IVP embryos carried out, and most of them were done in Russia
followed by the Netherlands, Spain, Germany, and France. The total number of embryo transfers (both of IVP and in
vivo-derived embryos) all over the world was over 1.5 million in 2017 .

IVF Made Easy:

Bovine IVP systems are working very well, but there is always room for improvement.
Approximately 90% of the aspirated oocytes reach MII stage, and some 80% of these
become fertilised in vitro. After culture in vitro around 50% of the fertilised oocytes become
blastocysts . The success story of the cattle IVP could have been very different due to the
appearance of the large offspring syndrome (LOS) in the late 1980s and the following
decade. After transfer of IVP embryos, alarming reports started to emerge, beginning in the
farming press, where some of the calves born had malformations and were too large, which
led to difficult calvings . Naturally, this became a serious problem for animal welfare, and
researchers searched for the background mechanism and a solution. Even at present, the
full mechanisms are unknown, but the addition of serum to the culture medium has been
identified as a likely factor behind the LOS in both sheep and cows.Today bovine serum
albumin and amino acids are used during culture, instead of serum, with better results. It
seems likely that the effects of the serum were through epigenetic changes . One of the
lessons to be learned from the LOS legacy is that seemingly minor changes in medium
composition may have a pronounced impact on the offspring. OPU has been modified for
successful use in cattle. The technique in cattle is supported by the possibility of the
operator to move an ovary to the right position via a gloved hand in the rectum of the cow.
It is the major technique to retrieve oocytes for IVP and ET .n the Swedish University of
Agricultural Sciences (SLU), Uppsala, extensive studies have been carried out to investigate
the animal welfare effects of repeated OPU sessions on heifers , the only negative
experience seemingly being the routine administration of the epidural anaesthesia needed.
Even though many techniques used in humans have worked very well in cattle, the ICSI
procedure is still far from successful, possibly because no spontaneous activation of the
oocyte occurs after injection of a spermatozoon . However, it could also be attributed to
other factors such as the darkness of the ooplasm, the large sperm heads, and the
toughness of the oolemma. This is in contrast to other forms of micromanipulation that
seem to work nicely in cattle. Micromanipulation of bovine embryos for sexing and/or
genotyping is becoming more and more common, with around 10,000 embryos tested in
2017 . The number is likely to have increased since 2017 when more relevant genetic
information for selection of the best possible traits in cattle rapidly became available. This
possibility to make fast genetic progress also increases the use of IVP in cattle.
IVP in pigs
The production of pig embryos in vitro still has many problems to overcome before it can be
said to be efficient. One of the main problems is inherent to IVF itself, which often results in
polyspermic fertilisation. When the sperm concentration is decreased, there is just a minor
effect until the concentrations are so low that there will be no penetration of spermatozoa at
all. A failure of the pig oocyte maturation in vitro is probably connected to the polyspermic
situation), but other factors such as an altered zona reaction , and even temperature during
IVF , have been proposed. The pig IVP has a two-day maturation process, and it is likely that
there are many conditions having an impact on oocyte maturation that we are not aware of as
yet. IVM in pigs gives relatively high rates of oocytes matured to MII stage, between 75%
and 85%, but unfortunately the polyspermic rates can reach 50–70%. This was observed in
the first experiment using single-layer colloid centrifugation before pig IVF was performed in
the IVP unit at SLU, Uppsala (. To overcome the extreme fertilising efficiency of the colloid
centrifugation-selected spermatozoa, the concentrations of spermatozoa for fertilisation had
to be lowered from the standard 5X105 spermatozoa/mL to 3X105spermatozoa/ mL. Since
polyspermic porcine embryos can cleave and develop to the blastocyst stage, as normal
embryos , polyspermia has to be assessed by investigation of pronuclei. Summary of IVF in
common domestic mammals. Species Main purpose of IVF Approximated number of in
vitro-produced embryos in 2017 Examples of specific characteristics Bovines Genetic
progress 1,000,000a Most techniques used in humans work well except ICSI Porcines
Research and genetic modifications Limited number and not possible to estimate Prone to
polyspermia in vitro, and gametes are highly sensitive to low temperatures Felines Model for
endangered feline species Very limited number and not possible to estimate Induced
ovulators Equine Increasing reproductive potential among selected individuals 500a IVF is
not successful, but ICSI and cloning are performed Canine Research Extremely limited
number and not possible to estimate Oocytes are in MI stage at ovulation and difficult to
mature in vitro Ovine Genetic progress 100a Surgical ovum pick-up and transfer are needed
aReference. UPSALA JOURNAL OF MEDICAL SCIENCES 71 This has to be done via
staining or high-g centrifugation in most domestic animals since the ooplasm is too dark to
visualise the pronuclei in light microscopy. If the oocytes are monospermic, around 80% can
reach the blastocyst stage . Recent research within this field is mostly focussed on selecting
spermatozoa in various ways to avoid polyspermy (50). ICSI is used with some success in
pigs , thereby bypassing the polyspermy problem entirely. Apart from the polyspermy
problem, pig oocytes and embryos are very sensitive to low temperatures. If temperature
drops below 25 oC the oocyte maturation process permanently stops in most cases With pig
embryos, it is not possible to store them at a low temperature before transfer since they are all
irreversibly damaged if temperature drops below 10 oC . Furthermore, porcine oocytes and
embryos, compared with other mammalian species, are very sensitive to cryopreservation,
possibly due to the high amount of lipids present . The advantages of an efficient pig IVP are
many. The genetical and technical progress could of course be significantly secured and
enhanced, but the pig also has a high value as a model for human situations. Despite many
difficulties, IVP in pigs is continuously explored. Research in Uppsala led to the first
production of piglets after fertilisation and culture in chemically defined media where the
effects of various components.

IVP in cats
No doubt, the domestic cat population as a whole has no problem to reproduce. However, the
domestic cat is an excellent model for other feline species, and almost all large cats are
threatened by extinction at least to some extent. Only a few domestic kittens have been born
after IVF . When a genetically important animal unexpectedly dies, gametes can be collected
and either directly subjected to IVP or frozen for future use. For cats, between 40% and 80%
of the COCs mature to MII phase, and the proportion of cleaved embryos developing in vitro
can reach 70% but is usually lower. In SLU Uppsala, we have used the domestic cat as a
model to increase the number of spermatozoa that can be salvaged from the testicles after
removal. The standard procedure is to collect only spermatozoa from the cauda epididymis
since this is where the most mature spermatozoa can be found . Using an IVP system, we
found that spermatozoa from the corpus epididymis had fertilising capacity and blastocysts
were produced, even if it was at a lower rate . With this feline model we saw around 8%
parthogenetically activated oocytes, but none continued to the blastocyst stage (unpublished
data). Since the parthogenic embryos can be similar to normal embryos, they cannot be
separated by simple microscopy.

IVP in horses, dogs, and sheep

Even though this short review cannot cover all domestic mammals where IVF has been performed or
attempted, some common species have to be mentioned. IVF methodology in the horse has so far never
been well established, but ICSI and cloning have been done with good results . The reason for the failed
IVF in the horse has been extensively . and is believed to be due to the failure to capacitate stallion
spermatozoa. The interest for IVF and related technologies for the horse is increasing within certain sports
and breeds where money is available and reproductive biotechnologies are allowed. Unfortunately, mares
seem to be poor responders to superovulation , and only one or few oocytes can commonly be retrieved per
cycle; this, together with the lower access to slaughterhouse ovaries, makes progress in this field slow. The
total number of in vitro-derived embryos transferred in the horse is currently low (Table 2). However, the
numbers can be expected to rise in the coming years as ICSI, and possibly also cloning, become more
established.

Dogs pose a special case since their oocytes are ovulated in the MI stage and several days are required for
them to mature to the MII stage in the oviduct. In vitro maturation has never been truly established, but
after in vivo maturation and in vivo culture the first—and, so far, only—puppies were born after IVF in
2015 . The main hurdles for dog IVP are the maturation process and the fact that dogs are monooestroual
breeders, which means that the oestrus is followed by a long pause, resulting in a bitch normally only
having one to three oestrous periods during one year. In vitro production of sheep embryos is well
established, but success rates are lower than for bovines .. Many factors regarding sheep IVP correlate well
to cows as they are both ruminants. Unfortunately, surgical OPU and transfer are still necessary in sheep
due to their anatomy, and this makes the procedure far more invasive than in bovines. The commercial
activity of ovine in vitro-derived embryo transfer is very low (Table 2).

Animals as models for humans

In many cases animals can be used as models for humans with regard to the final oocyte maturation,
fertilisation, and early embryo development in vitro. Depending on which factors are to be investigated,
different species might be of interest. Thus, rodents are not always the most suitable models for humans,
especially when considering oocyte maturation and fertilisation . One example is the oocyte transcriptome
where human oocytes are more closely related to cow oocytes than to mouse oocytes . There are also many
other specific characteristics making the human, the cow, and the pig much more similar compared with
rodents, for example the timing of the early embryo development in vitro .
In the IVP unit for pigs in SLU Uppsala, the impact of stress was investigated by adding plasma from
stressed or non-stressed sows to the IVP media . Plasma was collected from the corresponding time points
around ovulation in vivo, and stress was simulated by adrenocorticotropic hormone injections. In these
studies, stress seemed to have a major impact on spermatozoa function at the time of fertilisation, possibly
by prematurely induced acrosome reactions . In the SLU Uppsala IVP unit for cows, several studies have
been conducted with focus on the impact of insulin during IVM . These studies were mainly focussed on
the metabolic imbalance seen in dairy cattle after calving but are also of comparative interest to metabolic
disorders in humans. We observed a negative impact of insulin in IVM on blastocyst development,
evidenced by an effect on the gene expression patterns as well as morphologically. As a final example,
LOS should be mentioned. Since LOS can be induced by the addition of serum to culture medium, it has
been proposed as a model for Beckwith–Wiedemann syndrome in human . So, hopefully this major
setback can be made useful in future research.

In a nutshell, IVF is the process of collecting eggs and fertilizing them in the laboratory. The
resulting embryos are cultured for 3-5 days before they are transferred into the uterus or
frozen for future use.

IVF is significantly more successful than natural fertility due to the high number of eggs
produced in one cycle and the minimal number of sperm needed to fertilize them.
There are 5 basic steps in IVF: stimulation of the ovaries, egg retrieval, fertilization, embryo
culture and embryo transfer. Only the first step, ovarian stimulation, changes with the type of
patients. The rest of the IVF process stays the same. Let’s study each step:

1. Ovarian Stimulation:

Although a woman can produce 10-20 eggs every cycle, only one of them is allowed to grow
and ovulate in a natural cycle. To improve the chance of success with IVF, medications are
used to directly stimulate the ovaries to override this selective tendency in order to allow
more eggs to reach maturity. These stimulating medications contain FSH (follicle stimulating
hormone) to make multiple follicles grow.

However, the body has its own defense against multiple pregnancies. When facing multiple
growing follicles, the brain and pituitary can trigger ovulation of the largest follicle by
releasing LH (luteinizing hormone). Once the first egg has been released all the remaining
follicles will stop growing.

Thus, we must use another type of medication to prevent the interference of Mother Nature.
These medications (Lupron or a GnRH antagonist) act on the brain and pituitary to prevent
the release of LH to cause premature ovulation. Once the risk of premature ovulation has
been eliminated, ovarian stimulation can proceed until the follicles become mature and ready
for egg retrieval.
Once the pituitary can no longer interfere with the growth of the follicles, stimulation of the
ovaries can begin with close monitoring of the ovaries by ultrasound and hormone levels.
This is where there can be great variation as patients can benefit from different stimulation
protocols depending on their unique situations.

Choosing the correct stimulation protocol is one of the most important decisions in IVF
planning. At IVFMD we take great care in assessing every patient’s situation when designing
the ovarian stimulation protocol. The reserve of the ovaries, as determined by the AMH level
and the antral follicle count, plays a critical role in helping us decide the stimulation protocol.
Patients who have normal egg reserve can use the regular IVF protocols, whereas patients
with low egg reserve can benefit from one of the aggressive protocols.

2. Egg Retrieval

After about 10 days of ovarian stimulation with injectable medications, most of the follicles
should mature to size. An injection called HCG (Human Chorionic Gonadotropin) is
administered to induce the final maturation of the eggs.

About 36 hours after HCG triggering, the eggs are aspirated through the vagina under
ultrasound guidance in the office. The patient sleeps for about 20 minutes under deep IV
sedation given by an anesthesiologist. Once the retrieval is complete the patient wakes up
almost immediately after the IV medications are discontinued. Most women have mild pelvic
soreness and cramping after the procedure but this is self limited, resolving within a couple of
hours after the retrieval.

For safety reasons our anesthesiologists require patients to be at body mass index (BMI) of
38 or lower. High body mass can allow accumulation of the anesthetic Propofol that in high
dose can suppress respiration.

Once the eggs are collected, identified and prepared, they are ready for fertilization in the lab.
3. Fertilization

After their aspiration, the eggs are isolated under the microscope and are fertilized the same
day. We have two methods of fertilization, IVF and ICSI. In conventional IVF each egg is
incubated overnight in a droplet of special media with 70,000 motile sperm. Nature will
determine the best sperm to fertilize the egg.

In cases of low sperm concentration or motility, ICSI (Intracytoplasmic Sperm Injection) is

required to ensure fertilization. Under the microscope, one normal appearing sperm is chosen
and injected directly into the egg.

The day after the eggs are inseminated or sperm injected they are checked for signs of
fertilization. An egg is considered to fertilize normally when 2 pronuclei (one from each
parent) are seen. The average rate of normal fertilization by IVF or ICSI is 70%. The day
after the retrieval we check for fertilization. Typically, about 70% of the mature eggs are
fertilized.

4. Embryo Culture
The fertilized eggs are cultured for 5 days, which is the same number of days an embryo
takes to travel from the distal end of the tube into the uterus during natural conception. The
embryos are cultured in very special conditions where the temperature, gases and pH are
precisely controlled. In order to minimize fluctuations in the air and temperature of the
incubator, we check the embryos on day 1, 3 and 5, counting day 1 as the day after retrieval.
Patients are informed of the progress of their embryos after each check.

During this period the woman takes progesterone to prepare her uterine lining for the embryo
transfer.

5. Embryo Transfer

The ability to transfer well is one of the most critical factors contributing to the cycle
outcome. At IVFMD most embryo transfers are conducted on day 5 of culture. The transfer
procedure is painless and does not require sedation or anesthesia.

One or two embryos with the best grading scores are loaded into a soft catheter and delivered
into the uterus at a depth previously determined during the trial transfer visit. In difficult
cases, ultrasound guidance is used to assist the transfer. The remaining embryos are frozen
for future use if they meet freezing criteria.

Below is a visual summary of the IVF process.

Below is a nice video summary of the IVF process.
IVF (In vitro fertilisation) is a type of assisted reproductive technology (ARTs) used
for infertility treatment and gestational surrogacy. A fertilized egg may be implanted into a
surrogate’s uterus, and the resulting child is genetically unrelated to the surrogate. In a
normal pregnancy, a male sperm penetrates a woman’s egg and fertilizes it inside her body
after ovulation, when a mature egg has been released from the ovaries. The fertilized egg then
attaches itself to the wall of the uterus, or womb, and begins developing into a baby. This is
known as natural conception. However, if natural or unassisted conception is not possible,
fertility treatment is an option. IVF has been used since the late 1970s. On 25 July 1978, the
first “test-tube baby,” Louise Brown, was born.
THERE ARE FIVE BASIC STEPS FOR IN VITRO FERTILISATION:
STEP 1: SUPEROVULATION
 Medicines, called fertility drugs, are given to the woman to boost egg production.
 Normally, a woman produces one egg per month. Fertility drugs tell the ovaries to
produce several eggs.
STEP 2: EGG RETRIEVAL AND MATURATION
 A minor surgery, called follicular aspiration or ovum pickup, is done to remove the
eggs from the woman’s body.
 Using ultrasound images as a guide, the health care provider inserts a thin needle
through the vagina and into the ovary and follicles containing the eggs. The needle is
connected to a suction device, which pulls the eggs and fluid out of each follicle, one
at a time.
 In rare cases, a pelvic laparoscopy may be needed to remove the eggs. If a woman
does not or cannot produce any eggs, donated eggs may be used.
 The collected oocytes are matured in vitro under CO2 Incubator.
STEP 3: INSEMINATION AND FERTILIZATION
 The man’s sperm is processed and placed together with the best quality eggs. The
mixing of the sperm and egg is called insemination.
 Eggs and sperm are then stored in an environmentally controlled chamber called
CO2 Incubator. The sperm most often enters (fertilizes) an egg a few hours after
insemination.
 Sometimes the sperm is directly injected into the egg. This is known as Intra-
cytoplasmic Sperm Injection (ICSI).
STEP 4: EMBRYO CULTURE
 When the fertilized egg divides into 2 cells, 4 cells, 8-16 cells stage, it becomes an
embryo. Within about 5 days, a normal embryo has several cells known as morula and
blastocysts inside the CO2 Incubator.
STEP 5: EMBRYO TRANSFER
 Transfer of cultured embryos into the woman’s womb 3 to 5 days after egg retrieval
and fertilization is known as embryo transfer.
 The procedure is done in the doctor’s office while the woman is awake. The doctor
inserts a thin tube (catheter) containing the embryos into the woman’s vagina up into
the womb. If an embryo sticks to (implants) in the lining of the womb and grows,
pregnancy results.
  More than one embryo may be placed into the womb at the same time, which can
lead to twins, triplets, or more. The exact number of embryos transferred is a complex
issue that depends on many factors, especially the woman’s age.
 Unused embryos may be frozen and implanted or donated at a later date.
ADVANTAGES OF IVF

Nowadays infertility in male and female both is a major concern in the world, many factors
involved for this may be due to lifestyle, adulterated food, smoking, medicines and many
more. In vitro fertilisation helps many patients who would otherwise be unable to conceive.
The ultimate advantage of IVF is achieving a successful pregnancy and a healthy
baby. IVF can make this a reality for people who are infertile or unable to have a baby with
some of the following problems:
Blocked tubes: For women with blocked or damaged fallopian tubes, In vitro
fertilisation provides the best opportunity of having a child using their eggs.
Patients with a low ovarian reserve: IVF can be used to maximize the chance of older and
low ovarian reserve patients conceiving.
Male infertility: Couples with a male infertility problem will have a much higher chance of
conceiving with IVF than conceiving naturally.
PCOS: Polycystic ovary syndrome is a common condition in which there is a hormone
imbalance leading to irregular menstrual cycles. In vitro fertilisation has proved very
successful in patients with PCOS, who will not conceive with ovulation induction.
Endometriosis: Patients with endometriosis, where parts of the womb lining grow outside
the womb, IVF has proved very successful.
Premature ovarian failure: Women with premature ovarian failure or menopause can
have IVF treatment using donor eggs, which typically has high success rates.

In vitro fertilizationOpen pop-up dialog box

In vitro fertilization (IVF) is a complex series of procedures used to help with fertility or
prevent genetic problems and assist with the conception of a child.

During IVF, mature eggs are collected (retrieved) from ovaries and fertilized by sperm in a
lab. Then the fertilized egg (embryo) or eggs (embryos) are transferred to a uterus. One full
cycle of IVF takes about three weeks. Sometimes these steps are split into different parts and
the process can take longer.

IVF is the most effective form of assisted reproductive technology. The procedure can be
done using your own eggs and your partner's sperm. Or IVF may involve eggs, sperm or
embryos from a known or anonymous donor. In some cases, a gestational carrier — a woman
who has an embryo implanted in her uterus — might be used.
Your chances of having a healthy baby using IVF depend on many factors, such as your age
and the cause of infertility. In addition, IVF can be time-consuming, expensive and invasive.
If more than one embryo is transferred to your uterus, IVF can result in a pregnancy with
more than one fetus (multiple pregnancy).

Your doctor can help you understand how IVF works, the potential risks and whether this
method of treating infertility is right for you.

Why it's done

In vitro fertilization (IVF) is a treatment for infertility or genetic problems. If IVF is

performed to treat infertility, you and your partner might be able to try less-invasive
treatment options before attempting IVF, including fertility drugs to increase production of
eggs or intrauterine insemination — a procedure in which sperm are placed directly in your
uterus near the time of ovulation.

Sometimes, IVF is offered as a primary treatment for infertility in women over age
40. IVF can also be done if you have certain health conditions. For example, IVF may be an
option if you or your partner has:

 Fallopian tube damage or blockage. Fallopian tube damage or blockage makes

it difficult for an egg to be fertilized or for an embryo to travel to the uterus.

 Ovulation disorders. If ovulation is infrequent or absent, fewer eggs are

available for fertilization.

 Endometriosis. Endometriosis occurs when the uterine tissue implants and

grows outside of the uterus — often affecting the function of the ovaries, uterus
and fallopian tubes.

 Uterine fibroids. Fibroids are benign tumors in the wall of the uterus and are
common in women in their 30s and 40s. Fibroids can interfere with implantation
of the fertilized egg.

 Previous tubal sterilization or removal. If you've had tubal ligation — a type

of sterilization in which your fallopian tubes are cut or blocked to permanently
prevent pregnancy — and want to conceive, IVF may be an alternative to tubal
ligation reversal.
  Impaired sperm production or function. Below-average sperm concentration,
weak movement of sperm (poor mobility), or abnormalities in sperm size and
shape can make it difficult for sperm to fertilize an egg. If semen abnormalities
are found, your partner might need to see a specialist to determine if there are
correctable problems or underlying health concerns.

 Unexplained infertility. Unexplained infertility means no cause of infertility

has been found despite evaluation for common causes.

 A genetic disorder. If you or your partner is at risk of passing on a genetic

disorder to your child, you may be candidates for preimplantation genetic testing
— a procedure that involves IVF. After the eggs are harvested and fertilized,
they're screened for certain genetic problems, although not all genetic problems
can be found. Embryos that don't contain identified problems can be transferred
to the uterus.

 Fertility preservation for cancer or other health conditions. If you're about

to start cancer treatment — such as radiation or chemotherapy — that could
harm your fertility, IVF for fertility preservation may be an option. Women can
have eggs harvested from their ovaries and frozen in an unfertilized state for
later use. Or the eggs can be fertilized and frozen as embryos for future use.

Women who don't have a functional uterus or for whom pregnancy poses a
serious health risk might choose IVF using another person to carry the
pregnancy (gestational carrier). In this case, the woman's eggs are fertilized with
sperm, but the resulting embryos are placed in the gestational carrier's uterus.
Risks

Risks of IVF include:

 Multiple births. IVF increases the risk of multiple births if more than one
embryo is transferred to your uterus. A pregnancy with multiple fetuses carries a
higher risk of early labor and low birth weight than pregnancy with a single
fetus does.

 Premature delivery and low birth weight. Research suggests that IVF slightly
increases the risk that the baby will be born early or with a low birth weight.

 Ovarian hyperstimulation syndrome. Use of injectable fertility drugs, such as

human chorionic gonadotropin (HCG), to induce ovulation can cause ovarian
hyperstimulation syndrome, in which your ovaries become swollen and painful.
 Symptoms typically last a week and include mild abdominal pain, bloating,
nausea, vomiting and diarrhea. If you become pregnant, however, your
symptoms might last several weeks. Rarely, it's possible to develop a more
severe form of ovarian hyperstimulation syndrome that can also cause rapid
weight gain and shortness of breath.

 Miscarriage. The rate of miscarriage for women who conceive using IVF with
fresh embryos is similar to that of women who conceive naturally — about 15%
to 25% — but the rate increases with maternal age.

 Egg-retrieval procedure complications. Use of an aspirating needle to collect

eggs could possibly cause bleeding, infection or damage to the bowel, bladder or
a blood vessel. Risks are also associated with sedation and general anesthesia, if
used.

 Ectopic pregnancy. About 2% to 5% of women who use IVF will have an

ectopic pregnancy — when the fertilized egg implants outside the uterus,
usually in a fallopian tube. The fertilized egg can't survive outside the uterus,
and there's no way to continue the pregnancy.

 Birth defects. The age of the mother is the primary risk factor in the
development of birth defects, no matter how the child is conceived. More
research is needed to determine whether babies conceived using IVF might be at
increased risk of certain birth defects.

 Cancer. Although some early studies suggested there may be a link between
certain medications used to stimulate egg growth and the development of a
specific type of ovarian tumor, more-recent studies do not support these
findings. There does not appear to be a significantly increased risk of breast,
endometrial, cervical or ovarian cancer after IVF.

 Stress. Use of IVF can be financially, physically and emotionally draining.

Support from counselors, family and friends can help you and your partner
through the ups and downs of infertility treatment.
How you prepare

The Centers for Disease Control and Prevention and the Society for Assisted Reproductive
Technology provide information online about U.S. clinics' individual pregnancy and live
birth rates.

When choosing an in vitro fertilization (IVF) clinic, keep in mind that a clinic's success rate
depends on many factors, such as patients' ages and medical issues, as well as the clinic's
treatment population and treatment approaches. Ask for detailed information about the costs
associated with each step of the procedure.

Before beginning a cycle of IVF using your own eggs and sperm, you and your partner will
likely need various screenings, including:

 Ovarian reserve testing. To determine the quantity and quality of your eggs,
your doctor might test the concentration of follicle-stimulating hormone (FSH),
estradiol (estrogen) and anti-mullerian hormone in your blood during the first
few days of your menstrual cycle. Test results, often used together with an
ultrasound of your ovaries, can help predict how your ovaries will respond to
fertility medication.

 Semen analysis. If not done as part of your initial fertility evaluation, your
doctor will conduct a semen analysis shortly before the start of an IVF treatment
cycle.

 Infectious disease screening. You and your partner will both be screened for
infectious diseases, including HIV.

 Practice (mock) embryo transfer. Your doctor might conduct a mock embryo
transfer to determine the depth of your uterine cavity and the technique most
likely to successfully place the embryos into your uterus.

 Uterine exam. Your doctor will examine the inside lining of the uterus before
you start IVF. This might involve a sonohysterography — in which fluid is
injected through the cervix into your uterus — and an ultrasound to create
images of your uterine cavity. Or it might include a hysteroscopy — in which a
thin, flexible, lighted telescope (hysteroscope) is inserted through your vagina
and cervix into your uterus.

Before beginning a cycle of IVF, consider important questions, including:

 How many embryos will be transferred? The number of embryos transferred

is typically based on age and number of eggs retrieved. Since the rate of
implantation is lower for older women, more embryos are usually transferred —
except for women using donor eggs or genetically tested embryos.

Most doctors follow specific guidelines to prevent a higher order multiple

pregnancy — triplets or more — and in some countries, legislation limits the
number of embryos that can be transferred. Make sure you and your doctor
agree on the number of embryos that will be transferred before the transfer
procedure.
  What will you do with any extra embryos? Extra embryos can be frozen and
stored for future use for several years. Not all embryos will survive the freezing
and thawing process, although most will.

Cryopreservation can make future cycles of IVF less expensive and less
invasive. Or, you might be able to donate unused frozen embryos to another
couple or a research facility. You might also choose to discard unused embryos.

 How will you handle a multiple pregnancy? If more than one embryo is
transferred to your uterus, IVF can result in a multiple pregnancy — which
poses health risks for you and your babies. In some cases, fetal reduction can be
used to help a woman deliver fewer babies with lower health risks. Pursuing
fetal reduction, however, is a major decision with ethical, emotional and
psychological consequences.

 Have you considered the potential complications associated with using

donor eggs, sperm or embryos, or a gestational carrier? A trained counselor
with expertise in donor issues can help you understand the concerns, such as the
legal rights of the donor. You may also need an attorney to file court papers to
help you become legal parents of an implanted embryo.
What you can expect

Egg retrieval techniqueOpen pop-up dialog box

ICSIOpen pop-up dialog box
BlastocystOpen pop-up dialog box

IVF involves several steps — ovarian stimulation, egg retrieval, sperm retrieval, fertilization
and embryo transfer. One cycle of IVF can take about two to three weeks, and more than one
cycle may be required.

Ovulation induction

If you're using your own eggs during IVF, at the start of a cycle you'll begin treatment with
synthetic hormones to stimulate your ovaries to produce multiple eggs — rather than the
single egg that normally develops each month. Multiple eggs are needed because some eggs
won't fertilize or develop normally after fertilization.

You may need several different medications, such as:

  Medications for ovarian stimulation. To stimulate your ovaries, you might
receive an injectable medication containing a follicle-stimulating hormone
(FSH), a luteinizing hormone (LH) or a combination of both. These medications
stimulate more than one egg to develop at a time.

 Medications for oocyte maturation. When the follicles are ready for egg
retrieval — generally after eight to 14 days — you will take human chorionic
gonadotropin (HCG) or other medications to help the eggs mature.

 Medications to prevent premature ovulation. These medications prevent your

body from releasing the developing eggs too soon.

 Medications to prepare the lining of your uterus. On the day of egg retrieval
or at the time of embryo transfer, your doctor might recommend that you begin
taking progesterone supplements to make the lining of your uterus more
receptive to implantation.

Your doctor will work with you to determine which medications to use and when to use
them.

Typically, you'll need one to two weeks of ovarian stimulation before your eggs are ready for
retrieval. To determine when the eggs are ready for collection, your doctor will likely
perform:

 Vaginal ultrasound, an imaging exam of your ovaries to monitor the

development of follicles — fluid-filled ovarian sacs where eggs mature

 Blood tests, to measure your response to ovarian stimulation medications —

estrogen levels typically increase as follicles develop, and progesterone levels
remain low until after ovulation

Sometimes IVF cycles need to be canceled before egg retrieval for one of these reasons:

 Inadequate number of follicles developing

 Premature ovulation

 Too many follicles developing, creating a risk of ovarian hyperstimulation

syndrome

 Other medical issues

If your cycle is canceled, your doctor might recommend changing medications or their doses
to promote a better response during future IVF cycles. Or you may be advised that you need
an egg donor.
Egg retrieval

Egg retrieval can be done in your doctor's office or a clinic 34 to 36 hours after the final
injection and before ovulation.

 During egg retrieval, you'll be sedated and given pain medication.

 Transvaginal ultrasound aspiration is the usual retrieval method. An ultrasound

probe is inserted into your vagina to identify follicles. Then a thin needle is
inserted into an ultrasound guide to go through the vagina and into the follicles
to retrieve the eggs.

 If your ovaries aren't accessible through transvaginal ultrasound, an abdominal

ultrasound may be used to guide the needle.

 The eggs are removed from the follicles through a needle connected to a suction
device. Multiple eggs can be removed in about 20 minutes.

 After egg retrieval, you may experience cramping and feelings of fullness or
pressure.

 Mature eggs are placed in a nutritive liquid (culture medium) and incubated.
Eggs that appear healthy and mature will be mixed with sperm to attempt to
create embryos. However, not all eggs may be successfully fertilized.
Sperm retrieval

If you're using your partner's sperm, he'll provide a semen sample at your doctor's office or a
clinic through masturbation the morning of egg retrieval. Other methods, such as testicular
aspiration — the use of a needle or surgical procedure to extract sperm directly from the
testicle — are sometimes required. Donor sperm also can be used. Sperm are separated from
the semen fluid in the lab.

Fertilization

Fertilization can be attempted using two common methods:

 Conventional insemination. During conventional insemination, healthy sperm

and mature eggs are mixed and incubated overnight.

 Intracytoplasmic sperm injection (ICSI). In ICSI, a single healthy sperm is

injected directly into each mature egg. ICSI is often used when semen quality or
number is a problem or if fertilization attempts during prior IVF cycles failed.
In certain situations, your doctor may recommend other procedures before embryo transfer.

 Assisted hatching. About five to six days after fertilization, an embryo

"hatches" from its surrounding membrane (zona pellucida), allowing it to
implant into the lining of the uterus. If you're an older woman, or if you have
had multiple failed IVF attempts, your doctor might recommend assisted
hatching — a technique in which a hole is made in the zona pellucida just before
transfer to help the embryo hatch and implant. Assisted hatching is also useful
for eggs or embryos that have been previously frozen as the process can harden
the zona pellucida.

 Preimplantation genetic testing. Embryos are allowed to develop in the

incubator until they reach a stage where a small sample can be removed and
tested for specific genetic diseases or the correct number of chromosomes,
typically after five to six days of development. Embryos that don't contain
affected genes or chromosomes can be transferred to your uterus. While
preimplantation genetic testing can reduce the likelihood that a parent will pass
on a genetic problem, it can't eliminate the risk. Prenatal testing may still be
recommended.
Embryo transfer

Embryo transfer is done at your doctor's office or a clinic and usually takes place two to five
days after egg retrieval.

 You might be given a mild sedative. The procedure is usually painless, although
you might experience mild cramping.

 The doctor will insert a long, thin, flexible tube called a catheter into your
vagina, through your cervix and into your uterus.

 A syringe containing one or more embryos suspended in a small amount of fluid

is attached to the end of the catheter.

 Using the syringe, the doctor places the embryo or embryos into your uterus.

If successful, an embryo will implant in the lining of your uterus about six to 10 days after
egg retrieval.

After the procedure

After the embryo transfer, you can resume normal daily activities. However, your ovaries
may still be enlarged. Consider avoiding vigorous activity, which could cause discomfort.
Typical side effects include:

 Passing a small amount of clear or bloody fluid shortly after the procedure —
due to the swabbing of the cervix before the embryo transfer

 Breast tenderness due to high estrogen levels

 Mild bloating

 Mild cramping

 Constipation

If you develop moderate or severe pain after the embryo transfer, contact your doctor. He or
she will evaluate you for complications such as infection, twisting of an ovary (ovarian
torsion) and severe ovarian hyperstimulation syndrome.

Results

About 12 days to two weeks after egg retrieval, your doctor will test a sample of your blood
to detect whether you're pregnant.

 If you're pregnant, your doctor will refer you to an obstetrician or other

pregnancy specialist for prenatal care.

 If you're not pregnant, you'll stop taking progesterone and likely get your
period within a week. If you don't get your period or you have unusual bleeding,
contact your doctor. If you're interested in attempting another cycle of in vitro
fertilization (IVF), your doctor might suggest steps you can take to improve
your chances of getting pregnant through IVF.

The chances of giving birth to a healthy baby after using IVF depend on various factors,
including:

 Maternal age. The younger you are, the more likely you are to get pregnant and
give birth to a healthy baby using your own eggs during IVF. Women age 41
and older are often counseled to consider using donor eggs during IVF to
increase the chances of success.

 Embryo status. Transfer of embryos that are more developed is associated with
higher pregnancy rates compared with less-developed embryos (day two or
three). However, not all embryos survive the development process. Talk with
your doctor or other care provider about your specific situation.
 Reproductive history. Women who've previously given birth are more likely to
be able to get pregnant using IVF than are women who've never given birth.
Success rates are lower for women who've previously used IVF multiple times
but didn't get pregnant.

 Cause of infertility. Having a normal supply of eggs increases your chances of

being able to get pregnant using IVF. Women who have severe endometriosis
are less likely to be able to get pregnant using IVF than are women who have
unexplained infertility.

 Lifestyle factors. Women who smoke typically have fewer eggs retrieved
during IVF and may miscarry more often. Smoking can lower a woman's chance
of success using IVF by 50%. Obesity can decrease your chances of getting
pregnant and having a baby. Use of alcohol, recreational drugs, excessive
caffeine and certain medications also can be harmful.