Throughout History, fertility has always been considered as critical for the survival of a family, ethnic group or nation, so much so that religions and civilisations encouraged polygamy to achieve the aim of preserving their society. It is therefore unsurprising for people experiencing infertility to be singled-out and looked upon negatively, even nowadays.
However, there has clearly been a shift in modern societies with lifestyle choices leading to a significant increase in pregnancies later in life.
Delivering genetically normal babies at an advanced maternal age is therefore one of the biggest challenges facing today's fertility specialist the world over.
Another such great challenge is infertility in men, which has been overlooked for a long time due to the social stigma attached to it and the focus being traditionally on their counterpart, but this changed due to social and scientific progress.
The history of in-vitro fertilisation (IVF) and embryo transfer started with animals in the early 20th century well before it was established in humans. The first IVF baby "Louise Brown" was born in 1978 as a result of a collaboration between Patrick Steptoe and Robert Edwards, who has been awarded a Nobel Prize in 2010 in recognition of his efforts.
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Scientific progress in biology and in particular genetics has led to increasing research and development into fertilisation methods which would allow achieving more reliably successful outcome, i.e. have the maximum healthy normal babies with minimum effort and cost. This has led to dramatic progress in the assisted reproduction technology, which has been constantly embracing new treatments, advances in pharmaceuticals, diagnostic tests and micromanipulation techniques.
In the last twenty years, many techniques in the field of IVF have been discovered such as Pre-implantation diagnosis (PGD), Assisted Hatching (AH), cytoplasm transfer, GVT (Germinal vesicle transfer), PNT (pronuclear transfer) and SCNT (somatic cell nuclear transfer). In my view, two of the most significant breakthroughs in the field over the last twenty years have been Pre-implantation Genetic Diagnosis (PGD) and Intracytoplasmic Sperm Injection (ICSI) for male factor infertility, which will be described in detail here.
All these fertilisation methods undoubtedly lead to serious ethical, social and financial issues with the scientific world and society having to face controversial issues such as genetic selection, use of stem cells or the disposal of rejected embryos, as well as issues of cost and funding for IVF.
Looking at the future, PGD and ICSI are being constantly refined and perfected and still hold many promises.
It seems that the time is near when anyone would be able to "order" their own baby-on-demand, i.e. a screened "perfect" embryo made from cryogenically preserved eggs and sperm and transferred on the day with all the chipped monitoring and at a low cost.
Pre-implantation Genetic Diagnosis (PGD)
Pre-implantation Genetic Diagnosis (PGD) is the technique whereby IVF embryos are tested or screened for specific genetic conditions before implantation of the selected one. The type of screening depends upon the disorder being diagnosed.
The technique was first performed on a rabbit embryo in 1968 and was pioneered on humans by a group of researchers at Hammersmith Hospital (Franklin and Roberts 2006) who applied it successfully in 1990 on a sex selection case for the Duchenne dystrophy, which is an autosomal dominant male-affecting disorder. Professor Alan Handyside and Professor Robert Winston used PGD to select a female embryo thus ensuring that the genetic disease was not transmitted to the child.
Since PGD requires IVF for the egg retrieval, the service is generally provided to couples by IVF centres. The main benefits of the method are that it reduces the risks of an embryo having genetic or chromosome disorders as well as the risks of miscarriage due to such disorders. The parents can have peace of mind that their baby is not affected from the genetic disorder and they can avoid the horrible prospect of miscarriage or termination of pregnancy in case of finding out about an anomaly later in the pregnancy. So it has given hope to families with genetic disorders to be able to enjoy a pregnancy as normal as possible.
PGD has been performed successfully on a variety of cases, which can be separated into three groups.
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The first group would be of those who have inherited a disorder, such as single gene defects. In case of autosomal dominant diseases such as Marfan's, myotonic dystrophy and Huntington, there is a 50% that an affected parent will transmit his genetic condition to a child. For autosomal recessive disorders such as cystic fibrosis, sickle cell disease and beta thalassemia, if both parents are carrier, then there is a 25% risk to transmit the condition to the child, whereas if one parent is a carrier and the other is affected, there is 50% risk for a child to be affected. X-linked disorders such as fragile X and haemophilia A have the same risk as their autosomal counterpart (i.e. X-linked dominant has the same risk as autosomal dominant and X-linked recessive has the same risk as autosomal recessive) with the difference being that X-linked disorders affect one gender only whereas autosomal diseases affect the offspring irrespective of the gender (Harper, Delhanty and Handyside 2001). With such high risk disorders, PGD offers a real solution by minimising those risks.
Then the second group represents those with chromosomal structural abnormalities especially translocation, such as Robertsonian Translocation (Avery and Mhairi G. MacDonald 2005).
Finally, the third group encapsulates the rest of the cases where PGD is not very commonly used such as HLA typing, mitochondrial disorders and cancer predisposition.
Once an embryo is obtained by IVF, it can then be tested for PGD using currently one of three techniques. The first one is a lab-based genetic analysis which is less invasive. Depending on what condition needs to be tested, this can consist of running a PCR (polymerase chain reaction) to check if the embryo's gene is normal or abnormal, a fluorescent in situ hybridisation (FISH) test to verify if a set of embryo chromosomes is normal but it can test only 9 or 12 set of chromosomes (John A. Collins 2007), or more recently CGH (comparative genome hybridisation) to carry out a full analysis of all sets of chromosomes (Wilton 2002).
The second class of technique is rather invasive and involves biopsy at different stages of embryo formation which includes polar body biopsy, cleavage stage embryo biopsy and blastocyst biopsy. Polar body biopsy before and after the fertilisation as the majority (more than 90%) of aneuploidies are considered as a result of maternal meiotic division defect (Nicolaidis and Petersen 1998), and no issue of mosaicism with this method (Geraedts, et al. 2010). Initial study showed that biopsy of both polar bodies can detect aneuploidy up to 89% and a much larger study on this finding is underway (ESHRE 2010).
Cleavage stage biopsy is done at 8-cell stage of the embryo and then one or two cells are removed on day 3 after fertilisation. Birth rate is higher (over 37%) with single cell removal for this type of biopsy which also avoids multiple pregnancies (De Vos A 2009). Over 50 PGD centres only resort to this procedure for PGD. It can be carried using different means such as laser, mechanical or using Acid Tyrode solution which is not recommended for breaking out layer of embryo (Thornhill, et al. 2004).
Blastocyst biopsy is performed at day 5 after fertilisation when the blastocyst has two populations of cells, an outer and an inner mass of cells. Cells removed from the outer layer which later forms the placenta. Trophectoderm biopsy is conducted on day 5 or 6 blastocyst. In both these techniques mosaicism and allele dropout (unbalanced allele amplification) is still a potential issue.
In case of monogenic disorder, trophectoderm biopsy using laser at blastocyst stage is advantageous in PGD and it has shown higher implantation rate in a pilot study (Kokkali, et al. 2006) as use of laser can make the biopsy much more accurate, efficient with no effect on embryo development (Taylor, Gilchrist and al 2010).
PGD along with blastocyst transfer increases IVF outcome especially in recurrent IVF failures, as demonstrated by (Pehlivan, et al. 2003).
There is more drastic control over PGD centres which are regulated by ESHRE in the UK as compared to mere IVF centres.
PGD has been successfully applied to screen and avoid single-gene anomalies, but it is not good enough in its current form to check multiple genetic issues due to shortcomings in technology.
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Ethical issues and Genetic counselling
Due to the complicated nature of the IVF process and the risk of failure, however small, there is an undoubtedly a mental as well as financial pressure on the couple undergoing treatment.
Patient counselling is highly recommended by a qualified genetic counselor along with psychological assessment prior to the PGD (Thornhill, et al. 2004).
To address ethical issues, countries have setup national committees such as Human Fertilisation and Embryology Authority (HFEA) in the UK.
Pre-implantation Genetic Screening (PGS)
Pre-implantation genetic screening (PGS) is used for to check the accurate number of chromosome in embryo it is also called aneuploidy screening. It is more commonly used for old aged women, multiple IVF cycle failure or recurrent miscarriages to check if the embryo has a chromosomal abnormality (HFEA 2009). Like PGD, it also require for the couple to have IVF. According to the last HEFA study, over 3700 PGS tests were performed in 2007-2008 (Harper JC, 2010) and largest number of all the categories.it has proved to double the pregnancy rate especially for women aged more than 40 (Milán, et al. 2010) some have suggested it may increase pregnancy rate in AMA (Wilton 2002) while it is also considered to decrease pregnancy rate (Mastenbroek, et al. 2007) but on the other hand, eleven randomised controlled trials (RCT) showed no significant benefit using PGS for advanced maternal age (Harper, Coonen, et al. 2010) but a pilot study using polar body biopsy confirmed chromosomal status with 27 % implantation rate per embryo transfer. Further randomised controlled trial using much larger sample is underway (ESHRE 2010).
Future of PGD
The possibilities for improvement of PGD are vast, from technology, cost to lifelong disease prevention.
Use of laser is becoming very prevalent in IVF like in ICSI especially in patients with fragile oolemma who can benefit from laser assisted ICSI (Rienzi and al 2001). It can also be used for sperm immobilisation (Ebner and al 2001).
PGD only contributes a small percentage to prenatal diagnosis due its prerequisite to have IVF embryos for using the method. Some ideas could be developed to allow screening on all embryos (including non-IVF) by testing the maternal blood to retrieve fetal DNA samples. Currently, free fetal DNA is available but its use is very limited.
It seems very likely that PGD will be widely used for genome screening pre-IVF or pre-having children. It could be developed for other systemic chronic lifelong illnesses such as motor neuron disease or Parkinson's.
Growing embryo outside womb, reducing the risk of multiple pregnancies
PGD has been used for sickle cell anemia, which is a severe autosomal recessive disorder prevalent in Africa (Xu, et al. 1999). PGD should be used in adjunction to IVF in national programmes in places in Africa, but this could only be possible once the cost of the treatments is significantly reduced through research and development of new affordable methods for PGD.
PGD can be used to develop new cell lines for studying development of diseases and for cell replacement therapy, such as for myotonic dystrophy type 1 as described by (Mateizel, et al. 2006). This could be one possible area for groundbreaking findings.
The technique would need further technology progress to multiple gene defect detection.
PGD has the potential to save huge amounts of money which are currently spent on looking after severely handicapped children and it should be at least available to all the families with genetic disease if not nationally to everyone having IVF (Handyside 2010).
Initial fertilisation methods for male factor infertility such as Subzonal Sperm Insemination (SUZI), Partial Zonal Dissection (PZD) and direct injection of spermatozoa into the cytoplasm of the oocyte (DISCO) were discovered but were deemed unsatisfactory (Fishel, et al. 1993). SUZI provided a small success rate of 15% according to (Svalander, et al. 1994) for moderate male-factor infertility. There was still no solution for severe male infertility until 1992 with Palermo's findings.
The ICSI technique revolutionised the field of IVF especially for patients with male factor infertility when it was developed by Palermo for a patient who did not conceive following IVF and subzonal insemination of the oocyte (Palermo, Joris, et al. 1992).
According to the HEFA, over 12,000 babies were born using IVF and ICSI between 1992 and 2006 and it shows that at the beginning of using ICSI, the birth rate was lower than IVF but since 1995 it has been improving 2-3% as compared to IVF, and by 2006 birth rate was 30% with ICSI as compared to 27% with IVF.
The technique involve the direct injection of the single sperm into the ooplasm during metaphase2.It can be used in almost all forms of male factor infertility (Palermo, Cohen, et al. 1995).
Causes of male factor infertility can be due to defect in sperm motility, shape and its transport from epididymis (storage and transport duct), vas deference tube that carry the sperm forward) or defect in seminiferous tubule(development site for spermatozoa).
A small percentage of patient will have azoospermia which could be due to obstruction in genital tract or congenital absence of vas deference(CABVD), or due to non-obstructive causes such as somniferous tubule failure also known as primary testicular failure, congenital conditions causing Klinefelter syndrome (i.e. sex chromosome aneuploidy) and Y chromosome deletion while the third cause of azoospermia is possibly due to hypothalamic-pituitary failure (Brinsden 2005).
The causes of male infertility should be explored as use of ICSI in the absence of male infertility factors has not demonstrated any benefits (Kim, et al. 2008).
According to WHO survey of causes of male factor infertility, about 50% of cases has no identifiable cause followed by just over 12% had varicocele and 11.2% had idiopathic oligospermia (i.e. low concentration of sperm) and only small percentage of cases had other causes like congenital, systemic, immunological and ejaculatory disorder and others (Bhattacharya and Hamilton 2006).
Sperm count has been the most important parameter to check male fertility so using WHO criteria which has recently been updated, considered semen vol 1.5 ml normal, total sperm number 39 million per ejaculate, motility (progressive and non progressive) 40%, normal morphological form 4%.(cooper et al 2009) but the criteria is poor indicator of assessment of sperm quality ( (Kini, et al. 2010), Irvine et al 1998, (Tomlinson, Kessopoulou and Barratt 1999).
Male subfertility should be investigated thoroughly as there are links between male subfertility and testicular cancer (Peng, et al. 2009).
Evaluating male subfertility at genetic level is important step towards the understanding of possible genetic defect in fertility mechanism which could be transmitted to the offspring in ICSI patients (Campbell and Irvine 2000).
Sperm can be extracted from epididymis or testis according to the type of azoospermia .There are several microsurgical techniques available for sperm retrieval in patients with azoospermia and technique will be selected depending upon the failure of sperm transport possibly due to the blockage in which case MESE (Microsurgical epididymal sperm aspiration) or failure of sperm production by testicles in which case TESE (testicular extraction of sperm) which can be used for freezing sperm to use in ICSI (Friedler, et al. 1997) in cases of non obstructive azoospermia and more recently frozen sperm has improved pregnancy rate (Kalsi, et al. 2010) and microsurgical TESE is considered a method of choice for sperm retrieval according to the meta-analysis done (Yang, et al. 2008).
According to the Cochrane Database 2008, in the absence of no preferred technique for sperm retrieval and they favoured the minimally invasive technique.
As the sperm morphology and concentration is the single most important parameter for good outcome in IVF in male infertility, the scientist in Israel developed a technique of selecting the most 'ideal' looking sperm using powerful microscopes and using it for ICSI and it is called intracytoplasmic morphologically selected sperm injection (IMSI) and new sperm morphology criteria developed called the motile sperm organelle morphology examination (MSOME) (Bartoov, Berkovitz, et al. 2002).
It has improved pregnancy rate (Bartoov, Berkovitz, et al. 2003) especially in cases of repeated unsuccessful ICSI cycle (Hazout, et al. 2006) and (Antinori, et al. 2008) as sperm with high DNA fragmentation and number of nuclear vacuole may have been the cause of ICSI failure (Berkovitz, Eltes and Yaari, et al. 2005) and (Berkovitz, Eltes and Ellenbogen, et al. 2006) which would have been selected otherwise in Standard ICSI. Furthermore, computer technology has been implemented to be more precise in sperm selection and Computer assisted sperm selection (MSOME) during ICSI increases implantation rates (Wilding, et al. 2010).
On the other hand time for sperm analysis, expertise of embryologist and cost of specialised equipment used in this technique has to be taken into account (Antinori, et al. 2008).
Many articles published showed link between ICSI and genetic anomalies and it is quite conceivable concept as ICSI seems to be a rather invasive procedure. Fact that individuals having ICSI have some kind of underlying defect in sperm production, activation or transport and mitochondrial DNA impact which can transmitted to the off spring makes it plausible that ICSI carries risk of chromosomal and sex aneuploidies (Bonduelle, et al. 2002).While some studies suggest association of birth defect with ICSI (Hansen, et al. 2005) and some consider it not statistically significant or not reliable (Lie, et al. 2005), (Van Steirteghem, et al. 2002) and on the other hand it is found rather beneficial as ICSI children may have more height than naturally conceived ones (Sutcliffe, et al. 2001).
In 2007, the ESHRE CAPRI workshop group acknowledged the possible underlying genetic defect in patients with severe azoospermia and advised Pre-implantation genetic diagnosis (PGD) and genetic counselling for the couple prior to the ICSI. Furthermore close follow up has been recommended due to the possible risk of low birthweight, prematurity, high perinatal mortality and urogenital anomalies in ICSI with a emphasis on long term post birth follow up of the ICSI children.
Advances in ICSI
Firstly, results of microinjection of spermatid in patients with severe non obstructive azoospermia in animal model have been successful (Ogonuki, et al. 2003) Spermatid can be used for ICSI (Fishel, Green and Bishop, et al. 1995).
Round spermatid and retrieved testicular spermatozoa have increased fertilisation by 24 and 79 % respectively (Fishel, et al. 1997) during testicular sperm retrieval if no success in finding spermatozoa then late spermatid can be used in ICSI and it can result in successful pregnancy (Mansour, et al. 2003). In vitro culture of round spermatid which later develop into late spermatid in a controlled medium can guarantee a fertilization (Cremades, Bernabeu and Barros 2001) but there has been reports of major anomalies in pregnancies using elongated spermatids in humans (Zech, et al. 2000).
Secondly, techniques to improve spermatogenesis in case of severe azoospermia which could be done by either reproducing normal spermatozoon using its genetic information or by stimulating spermatogenesis using embryonic stem cells (G. D. Palermo 2009) but gene expression should be taken into account
In vitro spermatogenesis could be an option in case of defect in germ cell that is responsible for sperm production. Spermatogenic failure could be partial where maturation damage in selective tubule (Silber, et al. 1997) which can be rescued by micro surgical technique (C. J. Silber 2000) or complete involving most tubules (Tsai, et al. 2000) .it can be caused by mainly by chromosomal aneuploidies like Klinefelter's syndrome, recurrent Y chromosome deletion and monogenic disorders like Kallmann syndrome (Visser and Repping 2010).
In some rare cases with congenital anomalies like absence of testis, nuclear transfer or cloning
In ICSI where the whole process of injecting a sperm with a needle and disrupting the oocyte membrane seems a step too far from natural process of fertilisation so an enzyme based receptor mediated process of sperm introduction into the oocyte might be an less invasive option in future as facilitating the ICSI process may be good but forcing it can cause damage.
To reduce the anomalies, ICSI should be coupled with Pre-implantation Genetic diagnosis.
The last twenty years have seen an exponential progress in the field of IVF with techniques such as PGD and ICSI. Despite some limitations, both of these techniques show promising results and will be refined to circumvent their current limits.
Research with the aim to improve minimally invasive and cost effective techniques with a high success rate and very low risk are the way forward.
Congenital Absence of Vas Deference
Comparative Genome Hybridisation
European Society of Human Reproduction and Embryology
Fluorescent In Situ Hybridisation
Human Fertilisation and Embryology Authority in the UK
Intracytoplasmic Sperm Injection
Intracytoplasmic morphologically selected sperm injection
Polymerase chain reaction technology
Pre-implantation Genetic Diagnosis
Pre-implantation Genetic Screening
Partial Zona Dissection
Subzonal Sperm Insemination