Reproduction is a fundamental feature of all life known. It is a basic drive in all animals including humans. Each individual organism exists because of reproduction. This basic drive which was once thought to be so very easy to be fulfilled has become a dream for many a couple. It is surprising and contradictory that in a nation like ours where there are continuous and tremendous measures taken by the government to control our population, there are couples who would like to do anything to have a child.
Majority of developing countries are experiencing a marked decline in Fertility rates for the past 40 years from 1960. Fertility declines were rare earlier and fertility rates actually rose in some countries between 1945 and 1960. Yet, many parts of the world with different cultural, political, economic and social backgrounds are experiencing low fertility rates from 1960.India is no exception to this trend.
The total fertility rate has declined from 6 in 1947 to 3.3 in 2000. It is expected to decline further to the level of replacement by 2020. A major contributor has been the increase in the average age at marriage. In 1961, the average age at marriage for men was 22 years and 16 for women. By 1993, this had increased to 26.5 and 24.5, respectively. There is a significant increase in the number of couples seeking medical treatment for infertility now more than ever before.
Infertility is defined as failure of conception after 12 months of uninterrupted sexual intercourse. One of the major factors determining this trend is that more and more women are delaying childbirth until their late 30's and into their 40's for various reasons, one of them being to develop their professional careers. This voluntary delay in childbearing not only poses a problem in terms of the 30-50% reduced pregnancy potential of older women, but also other risks like: the effect of pregnancy on other maternal illnesses, an increased risk of pre-eclampsia, hypertension and diabetes, and an increased risk of chromosomal abnormalities, abortions, and stillbirth.
The fact that decrease of female fecundity (ability to bear a child) begins in the early 30's, increases by mid thirties and becomes more pronounced after 40 is well documented. The most rapid decline in fertility potential in any single year has been found to occur at 35 years. Beyond this fertility is irreversibly lowered.
There is an approximately 70% decrease in the fertility rate of women attempting pregnancy at the age of 40 or older compared with younger women, and a twofold to threefold increase in the rate of spontaneous abortions. The biological basis of this reduced fecundity with increasing maternal age is due to various factors of which the effect of aging on every organ of the reproductive system is an all important factor.
Aging has an effect on every organ in the body and the ovary in the female is no exception. It is of general acceptance that most women are fertile through their early twenties. By mid thirties infertility rate increases by almost 30%. There is something known as the biological clock which is a direct result of the limited egg supply with which each woman is born.
Women are born with a finite number of germ cells and they are not replenished during their lifetime. Though this dogma has been challenged of late, there is no clear cut evidence so far to think otherwise. Before birth the female fetus has about 7 million follicles. Each follicle contains the oocyte or the egg, surrounded by granulosa cells or support cells for the oocyte. Of this initial number 2 million remain at birth. By puberty the follicular supply comes down to 300000. Of this pool, only 400 follicles are utilized during the women's lifetime for ovulation.
Every month, during the 28 day cycle, numerous follicles begin to develop out of which one dominant follicle matures and is ovulated or released. This utilization and attrition of the existing follicles from birth to menopause results in their reduced number with advancing age. In the 10-15 years before menopause there is a gradual acceleration in the follicular loss which is related to an increase in follicular stimulating hormone. More important than the reduction in the follicle number is the quality of oocytes which is a striking manifestation of ovarian aging.
In the human, 6 billion DNA molecules are located on 46 chromosomes which are coiled up inside the nucleus of each cell in our body and are divided into 23 pairs, 22 autosomal pairs and 1 sex chromosomal pair, the X and the Y. The child who has 2 X chromosomes will be a girl and one which has one X and one Y will become a boy.
For conception to occur, twenty-three chromosomes from the husband's set of forty-six, and twenty-three chromosomes from the wife's set of forty-six, must meet at the moment of fertilization and become an embryo with a new normal set of forty-six chromosomes. The process whereby primitive sperm cells and primitive eggs lose half of their chromosome number as they become sperm and mature eggs ready for fertilization is called "meiosis." The aging process of eggs makes it harder for them to undergo the meiosis process than sperm. That is why the eggs from older women are less likely to result in a viable embryo, and that is also why older women are more infertile than younger women, and why older women have higher rates of miscarriage and of babies with abnormalities such as Down's syndrome.
Oocyte quality in the female is an important determinant of successful fertilization. Some of the key contributing elements are oocyte maturation, spindle formation, energy supply, syngamy which is fusion of male and female pronuclei during the process of fertilization and early embryonic development. It has been proven scientifically that errors in some of these factors are more likely to happen with advancing age.
Other age related factors include irregular ovulation which occurs with increasing age as the hormone levels change, and luteal phase deficiencies which occur because too little progesterone - which is a hormone that maintains pregnancy in the uterus by making the uterine bed more receptive for the embryo to implant, is secreted. Increased miscarriages with aging as a consequence of altered chromosome numbers, called aneuploidy is also common in older women. This again is an important cause of decline in oocyte quality
Though there is no strong evidence that men suffer the same age related degeneration that women suffer, older sperm do cause genetic problems in the offspring like older eggs do. Unlike women, men are not born with spermatozoa in their testes. They start producing sperm at puberty and this process continues persistently throughout their life. Due to such high sperm production where the DNA is copied again and again, small variations or mutation occur in older men. These mutations cause a long list of genetic diseases in the offspring among which diseases like Lesch Nyhan Syndrome, Polycystic kidney disease and Hemophilia A are the most well known.
Age related changes also occur in the male both in the endocrine (hormonal) and sperm cell parameters. In the testis, the leydig cells which produce testosterone reduce in number and the aging pigment lipofuchin accumulates. There are changes also in the epidydimis, basal membranes and seminiferous tubules. There are localized changes in the spermatogenesis like degeneration leading to reduction in some types of spermatozoa, arrest of spermatogenesis at spermatocyte 1 stage and numerous malformations of spermatids. These changes are seen in relatively small areas and are diffusely distributed in the testis.
Along with genetic mutations, the semen volume, the percentage of motile sperm cells and the percentage of sperm cells with normal morphology also decline with age. Sperm motility and morphology are vital factors which determine the chances of conception. During normal intercourse the semen is deposited in the vagina and from here the spermatozoa in the semen have to swim up to the fallopian tubes of the female where a ripe egg is waiting (following ovulation) for fertilization to occur. Although these endocrine and sperm cell changes do not necessarily mean decline in fertility the incidence of sub fertility and time to achieve pregnancy increases when the male is aged more than 50. Also, for fathers over age 40, the risk of having a child with a disease-causing mutation is similar to the risk the mother has for a child with Down syndrome
The advent of assisted reproduction technologies has been able to overcome these problems and reduce the sufferings of infertile couples to some extent. During the past 20 years assisted reproduction has made great strides and we have advanced techniques to treat male and female infertility. Now older couples are also able to fulfill their desire to be parents through these techniques and even complicated issues like alarmingly low sperm counts in the male, ovarian and tubal defects in the female have been addressed by this evolving branch of medicine. But research has clearly shown that, success even with this most sophisticated technology is dependent on age. due to factors like decreased oocyte quality, spontaneous abortions and implantation failure as mentioned above. Embryo implanting ability and survival decline gradually after 30 years of age, but by more than two thirds after 40 years
It is evident from demographic studies that the mean age at motherhood has been increasing since 1980 with women delaying childbirth due to various reasons. The risk of childlessness increases with increasing age both in women and men. In women ovarian aging results in reduced number of eggs and impaired oocyte quality which is an important factor for good quality embryos to develop. The implantation potential of embryos decreases with increasing age as evidenced by increased spontaneous abortions in older women. In men advanced age causes reduced motility and morphology in sperms which affects normal conception. Both in men and women increased age causes genetic abnormalities in the quality of egg and sperm leading to the birth of children with genetic defects. Though much research needs to be done into the various factors affecting oocyte and sperm quality in older men and women, we are at a juncture to reevaluate our strategies and priorities in life for us to avoid the burden and displeasure of childlessness which cannot be helped beyond a certain extent even with the latest technical advances in science.
One of the difficult questions while deciding an embryo transfer is when to do the transfer. While some clinics only do Day 2 or Day 3 transfers, others prefer Day 5 or Blastocyst transfers. What is the difference and why the preference?
So should transfer at the blastocyst stage be the norm as some infertility specialists insist? What happens if a patient has only three quality embryos available on day 3? What is gained by delaying the transfer in this case? Blastocyst stage embryos have higher implantation potential when compared with cleavage stage embryos, since only those embryos with activated genome reach the blastocyst stage. This is reported to benefit IVF in (i) Improving pregnancy rates and (ii) reducing high incidence of multiple gestations, since only few embryos need to be transferred.
Blastocyst culture (day 5) has many advantages over early stage embryo transfer (day 2, day 3). In the female reproductive system, fertilisation of the egg and cleavage of embryos occur in the fallopian tube and the embryo implants in the uterine cavity at the blastocyst stage. Thus, there is a physiological synchronization of the blastocyst stage embryo with the endometrium. It is physiologically pre-mature to expose early stage embryos to the uterine environment. Also, studies have revealed that uterine contractions decreased significantly over time following HCG administration leading to a significant reduction in uterine pulsatility at the time when blastocysts are transferred and therefore chances that embryos can be expelled are less.
One important factor favouring blastocyst transfer is that a disturbingly large proportion of morphologically normal day 3 embryos are chromosomally abnormal, thus contributing to the 80-90% rate of implantation failure post transfer. While it is a point of debate that extended culture of embryos to day 5 cannot be used as a reliable tool to select against chromosome abnormalities, studies have clearly demonstrated that the incidence of abnormalities can be reduced from 59% to 35% for older women.
A major concern in blastocyst culture is that - less number of embryos on day 3, might lead to embryo loss during extended culture and cancellation of the cycle. The fear that difference between the in vitro and in vivo culture conditions might result in developmental arrest of otherwise viable embryos during extended culture, prevents many labs to go ahead with day 5 culture. Improvement in the culture media together with the optimization of laboratory conditions has enabled prolonged in vitro culture of embryos without compromising their viability. In our lab the overall blastocyst formation rate is 85.3% which has been achieved irrespective of the number of embryos on day 3.
In our experience, we have seen that the number of embryos available on day 3 may not necessarily act as the deciding factor for extended culture of embryos and good blastocyst formation and good pregnancy rates can be achieved with good laboratory conditions and technical expertise even in the presence of a single embryo, provided it is a good quality embryo.
The purpose of assisted hatching is to create a small opening in the zona of an embryo so that it may easily exit when it has developed sufficiently. This procedure was developed as a result of findings that egg and embryo zonae can physically harden or thicken when cultured in vitro. And despite normal development of the embryo, the in vitro-induced hardening characteristics of the zona might actually prevent the transferred embryo from implanting because of a failure to allow exit of the embryo. Assisted hatching can guarantee that an adequate opening will be available for the embryo to pass through when the time comes. Although some clinics require certain indications before performing AHA, we have found no evidence of any harmful effects associated with its use and therefore perform this procedure on all IVF embryos so that every couple could share in the benefits that we believe AHA provides.
AHA (Assisted hatching) is almost always performed on the day of the embryo transfer. The embryologist performs AHA on all embryos that are expected to be transferred, so if there are more embryos in culture than we wish to transfer, the embryologist selects those having the best quality for AHA. After all, we want our patients to have the best chance at pregnancy which means hatching and transferring the best-looking embryos. In the event that there are numerous high-quality embryos available for transfer on day 3, the embryologist will likely opt to delay transfer until the blastocyst stage which usually falls on day 5 or 6 after the retrieval (see day 3 transfer vs. blastocyst transfer discussion for criteria and reasons why one transfer may be selected over the other).
Before laser came into use for assisted hatching, a substance called acid tyrode was used to thin the outer covering of the embryos. This acid has to be released slowed through a pipette and the quantity released is directly proportional to the skill of the embryologist. With this technique the duration of exposure of the embryos to outside atmosphere was more thereby altering the Ph and this could prove detrimental to the embryo growth. With Laser technique the whole procedure takes only a few seconds and the embryos are put back in the incubator as quickly as 20 - 30 seconds after removing.
According to recent studies, the routine sperm analyses such as sperm count, motility and morphology, are not reliable indicators of sperm function.
Sperm chromatin is tightly packaged to protect DNA during the transit that occurs before fertilization. Any abnormality in chromatin structure and integrity could lead to failure in fertilization.
ICSI allows couples with very low sperm counts or poor quality sperm to achieve fertilization and pregnancy rates equal to traditional IVF. It is also recommended for couples who have not achieved fertilization in prior IVF attempts. Sperm of virtually any quality and from any level of the male reproductive tract may be used with the only criterion for use being that the sperm is alive even if it is not moving (motile).