Stillbirth, loss of a baby at delivery, is a painful challenge.  The suffering associated with the loss of a child, even before birth, can be overwhelming.  Especially acute for women that have conceived utilizing assisted reproduction, the loss of a pregnancy fought through reproductive technology can overwhelm a couple.  Stillbirth is a rare risk of pregnancy; the challenge facing us as reproductive medicine experts and obstetricians is how to reduce that risk.

The technologies of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) have enabled pregnancy for thousands of families with sperm, egg, and uterine problems.  With IVF, egg quality can be optimized using fertility drugs to produce more eggs.  Blocked fallopian tubes can be bypassed.  Weak sperm can achieve pregnancy by ICSI, where, using a microscopic needle, the sperm cell can be introduced into the egg.

No-one should expect these techniques to be foolproof.  While mechanical problems can be improved, other weaknesses in the reproductive system cannot.  Small deviations in the genetic code of the sperm or egg, missing chromosomes, aging, uterine defects, etc cannot be fixed by treating the sperm cell or embryo.

Thus the problem – these pregnancies established by high technology, are at higher risk.

A recent study from Denmark looked at stillbirth in children born after IVF/ICSI  and found that the risk was higher in children born after IVF/ICSI than natural pregnancy.  Out of 16,525 births to fertile women the chance of stillbirth was 0.37%, that is, 3.7 out of 1000 births.  Out of 742 babies born to women after IVF/ICSI there were 12 stillbirths, 1.62%, that is 16.2 out of 1000 births.

But more importantly to our patients, the liveborn baby rate after a successful IVF/ICSI treatment  and pregnancy is 98.4%.  The liveborn baby rate after a successful natural conception and pregnancy is 99.6%.  Almost all of the successful pregnancies after IVF/ICSI are liveborn.

Reproductive technologies, like IVF and ICSI, are enabling pregnancy and family building where it was not possible before.  All of our patients must be informed of and recognize the risks associated with fertility treatment.  These risks should not, however, dissuade anyone from considering these therapies.  On the contrary, the overwhelming likelihood is that, once a pregnancy is established, it will progress successfully to delivery and a healthy child.

We need to recognize these risks to provide help understand and take measures to reduce the risks to all children.  We will continue to watch these studies carefully in our ongoing effort to assure our patients of excellent pregnancy rates, at low risk.

Footnote:

1. K. Wisborg, H.J. Ingerslev, and T.B. Henriksen  IVF and stillbirth: a prospective follow-up study  Hum. Reprod. Advance Access published on February 23, 2010.

This past summer, Dr. Herbert and I had the opportunity to travel to Barcelona, Spain for the annual meeting of the European Society for Human Reproduction and Embryology (ESHRE). Though largely attended by Europeans, this scientific meeting draws physicians, embryologists and scientists from around the world to discuss their research, attend courses and lectures, and discuss the latest topics in our field.

Here are some of what I consider the highlights of the meeting:

Outcome of 1267 Children after Frozen Embryo Transfer – Study from Denmark

Control group: Fresh IVF pregnancies

Only 14% were twins

They compared 957 frozen embryo singletons with about 10,000 fresh IVF singletons

No increase in neurological problems or malignant diseases on FET babies.

No differences were seen when IVF or ICSI-derived frozen embryos were compared.

Results similar to prior Swedish study showing better outcomes for FET babies.

Why a better outcome? The authors postulated that patients conceiving with FET were more likely to be good prognosis patients.

Three years of clinical application in human oocyte vitrification (freezing): high survival rate and healthy deliveries (from Rome)

3138 unfertilized eggs were frozen between 10/04 – 10/07.

They reported on 295 cycles with planned embryo transfer – all patients were less than 40 years old. The patients underwent programmed endometrial preparation using a GnRH agonist (like Lupron) and oral estrogen and vaginal progesterone.

770 unfertilized eggs were thawed, 98.9% survived the thaw. The eggs were injected with sperm 2 hours after thawing and the embryos were transferred on Day 3.

Results: Avg. # embryos transferred = 2.3

Clinical pregnancy rate = 27.8%

Implantation rate = 13% per embryo, 11.3% per thawed egg. That is, about 11% of the eggs thawed resulted in a viable gestation.

58 deliveries of 63 babies, mean birth weight = 2930 grams

They experienced no congenital malformations at birth.

Then, the most controversial paper presented by Dr. Norbert Gleicher, an RE from New York.

The title: “In contrast to prevalent opinion, twin pregnancies after fertility treatments are medically, ethically and economically desirable outcomes.”

His arguments to support this opinion:

Most couples want to have more than one child. Therefore, they will need to undergo two pregnancies of two separate singletons vs. one pregnancy of twins to have two children. He argued that twins born after ART have much better pregnancy outcomes (by 30-50%) than spontaneously-conceived twins. He also argued that the accumulated costs and risks to mother and babies are higher with two singleton than one twin pregnancy.

Despite these intriguing arguments, this paper was hotly debated and essentially disavowed by the European ART community. Europe has led the way in legislating for avoidance of twins. In fact, in Denmark, if a woman has twins after the transfer of more than one embryo using IVF, she incurs any neonatal costs out of pocket.

Corifollitropin: a modification of Follistim to make it a once-a-week injection.

As most people know, the medication we most commonly use for fertility treatment, Follistim, is pure human FSH, manufactured using recombinant DNA technology. The company that makes Follistim, Schering Plough, is working towards FDA approval of a modified version of Follistim, called Corifollitropin, that will make the drug very long-acting. It may be possible to only take one injection per week!

A symposium at ESHRE presented information from studies underway in Europe and USA. Corifollitropin is not in clinical use yet, even in Europe, but will be very soon.

For those of you interested in the details, Corifollitropin is the recombinant FSH molecule + 22 C-terminal peptides from beta-hCG, It does not bind to the LH receptor.

This modification lengthens the half-life of Follistim from 22-34 hours to 60-74 hrs for Corifollitropin. After injection peak levels are reached in 2 days then slowly levels decline. The recommended regimen will be one dose per week, starting at baseline, switch to daily recombinant FSH after that.

	Carolyn Givens, M.D. was the first in San Francisco to successfully initiate a pregnancy using intracytoplasmic sperm injection (ICSI). She currently co-directs the Bay Area Pre-Implantation Genetic Diagnosis Program (PGD) and is director of PFC’s PGD program.
	Carl Herbert, M.D. was instrumental in the development of one of the first assisted reproductive technology programs in the United States and has been performing IVF longer than any physician in the Bay Area.

For many people, the dream of having a family also includes the dream of having children of both sex. Since most families today are much smaller than in generations past, the odds of having two or three or even four children of the same sex is fairly high.

Throughout human history, there always has been interest in methods to sway the chances of conceiving a child of a particular sex. Today, in the 21 st century, it is quite clear that many of these sometimes bizarre and sometimes simple home remedies have no basis in fact.

There are ways to significantly shift the odds of having a child of one sex or another. Sex is conferred on an embryo by whether an X-bearing sperm (for a girl) or a Y-bearing sperm (for a boy) enters the egg. Unfortunately, despite highly publicized claims, there are no proven effective “at home” methods of sperm separation. Nor does timing of intercourse relative to ovulation affect the 50:50 sex ratio. By natural methods, the ratio remains a flip of the coin.

The only commercially available method for sperm separation that appears to be effective is the sperm sorting process available through Microsort.net. This method involves using a fluorescent DNA dye that attaches to either X or Y chromosomes. The sperm then passes through a cell sorter that separates the sperm based on the fluorescence. This method is still under FDA investigation for safety and efficacy but does appear to do a reasonable job in separating sperm, especially if the desired sex is female.

Mirosort reports a 90% success rate with separating X-bearing sperm and a 73% success rate in separating Y-bearing sperm. There have been only a few hundred babies born thus far, but there does not appear to be any increase in birth defects. Because this process is still considered “experimental,” couples wishing to participate, will have to travel to either Fairfax, Virginia (Microsort headquarters) or an affiliated clinic in Southern California for fresh sperm insemination.

Unfortunately, after Microsort processing, the number of sperm available for insemination is severely decreased. Freezing and thawing of sperm, which would allow the sample to be shipped to another location, reduces these numbers even further. Because sperm counts are so low after sorting, it is usually necessary to do in vitro fertilization with sperm injection (IVF-ICSI) to significantly improve the fertilization in the IVF laboratory. PFC is a participating site in the FDA investigation for Microsort. We have used sperm specimens that had been previously Micro-sorted for IVF-ICSI.

Researchers at UC Irvine recently published a study describing the use of lasers to “trap” the heavier and slower moving X-bearing sperm to separate it from the lighter Y-bearing sperm. In the future, this process may provide an alternative to Microsort. However, it is not yet commercially available.

Beyond the Microsort technique, the only way to improve the odds of selecting one sex over another at close to 100% accuracy is to undergo Pre-Implantation Genetic Screening (PGS). PGS uses a DNA-binding technique to determine if there are a correct number of chromosomes in the embryo at the time of IVF. To complete this screening, embryos on Day 3 of culture (5-10 cells) undergo a biopsy to remove a single cell. The rest of the embryo remains in culture in the IVF laboratory. The removed cells are analyzed for the correct number of chromosomes. Currently, PFC with its cytogenetic partner, Genetics and IVF Institue screen embryos for 3-12 chromosomes. This screening is called “aneuploidy screening.” We allow our patients to know and select the sex of their normal embryos for transfer if they so wish.

Although IVF with PGS is the most effective method for sex selection, it is certainly the most expensive and there is no absolute guarantee that the transfer of the screened embryos will result in pregnancy. A PFC physician can best discuss the odds of success, based on the woman’s age and the couple’s history of childbirth.

Many couples undergoing PGS are doing so to screen for specific genetic defects or are specifically undergoing sex selection because of their risks of having a genetic disease that only affects males (X-linked diseases).

On the other hand, PGS for elective sex selection, either for “family balancing” or even for having a first child of a particular sex poses difficult ethical issues. Just because we have the ability to choose the sex of a child, should we? What will the couple do with normal embryos of the undesired sex? At PFC, we do not encourage PGS for elective sex selection. However, if a couple is undergoing IVF and wishes to undergo aneuploidy screening, we do allow them to select to transfer embryos by sex. We encourage all patients to consider donating excess embryos of the undesired sex for adoption by other couples.

Women or couples interested in this procedure should discuss it with their Reproductive Endocrinologist. At PFC, we also refer our PGS patients for a special genetic counseling session at California Pacific Medical Center in preparation for this process.

I had been trying to get pregnant for six months and didn’t want to wait any longer. In the past, my husband had gone through chemotherapy, but when we decided to begin our family, we never contemplated that his medical history would make conceiving a challenge.

Once we were ready to take the next step, our urologist recommended Pacific Fertility Center. Patients he referred to PFC had been successful, so we were very hopeful that they might be able to help us. We lived a distance from the center and had to make the 6-hour drive each way for treatments. We were determined to get the best care available.

Our cycle began way back in August of 2005. Initially we worked with Dr. Paul Turek, an urologist from UCSF in conjunction with PFC in cases like ours. He performed testicular mapping, looking for pockets of live sperm. Since only one pocket was found, Dr. Turek recommended my husband undergo FSH injections 3 months prior to our cycle, to increase sperm production. Fortunately, this experimental protocol worked better than expected and we were able to avoid invasive surgical removal of sperm. It only takes one good sperm to fertilize an egg and he was able to find more than enough.

In order to get my eggs to fertilize with my husband’s sperm, I went through IVF including FSH injections. The needles intimidated me, but I was able to get past that fear. Everything turned out OK and I made it through the procedure really well. During my retrieval they collected 20 eggs.

As it turns out, after fertilization with ICSI, we had 8 grade 1 and 2 embryos. Three of the embryos were transferred and the other five were frozen. To my pleasure, I became pregnant with a single baby girl. This was an amazing experience, especially considering the odds were not hugely in our favor. Once we got the good news, we were in an elated state of shock – we had been through a lot and finding out we were finally pregnant was wonderful news! I had an extremely easy and natural birth in May of the following year.

All in all the experience was quite a whirlwind; my husband and I had a lot of ups and downs. The assumption we had when we first decided to try to get pregnant was it would be natural and uncomplicated. However, learning we had an infertility problem was a devastating experience. What empowered my husband and me was that we started doing research about our problem. The more we learned, the more comfortable and less intimidated we felt. I would highly recommend this. In retrospect, the biggest ups and downs were when we got the reports on the good or the poor sperm samples. When they found sperm they could use, we could hardly contain our excitement.

I appreciate the care Dr. Isabelle Ryan provided. I liked her a lot and I have heard nothing but good things about all the PFC doctors. Dr. Ryan knows what she’s doing and was able to explain all the options available to us. Joe Conaghan, the lab director, was spectacular. He was able to find sperm in a sample that our local doctors could not. Being from a rural area, we didn’t have local access to PFC’s level of care and state-of-the-art technology. I absolutely trust PFC and have recommended them to others. If we decide to have another child, we will definitely come back. We love our little baby girl so much and every day is a new adventure. We can’t imagine our lives without her!

Susie & Steven D.

Male factor infertility is quite common, contributing to 40% of infertility diagnoses. Treatment is designed around the particular type of problem and can be remarkably effective. For those with male factor infertility, the initial course of action is to review personal health habits. Stress, poor diet, and alcohol use have all been correlated with male factor infertility. Alcohol use, in particular, has been shown to have a dose-related effect on sperm; the more one drinks, the poorer the reproductive outcome. High temperature exposure from hot tubs or hot baths (immersion in hot water), or heavy exercise, particularly bicycle riding, have been correlated with male factor infertility as well. Resting a laptop computer on one’s lap has also been implicated in raising testicular temperature.

Diagnosis of male factor infertility starts with a semen analysis. The semen analysis should be performed on an ejaculated sample collected on at least two occasions 2-7 days following abstention from sexual activity. Measurement of the sperm count, motility, and volume can reveal production problems as insufficient or poor quality sperm are released from the testes. Table 1 lists the standards for assessing a semen analysis (Source: The World Health Organization, 1992).

Additional tests to evaluate sperm quality include the detailed or Krueger morphology. This entails viewing individual sperm cells under a high-powered microscope. This is a strict test that reveals abnormalities in the shape and size of the sperm heads, mid-pieces, and tails. A normal morphology is present when over 14% of sperm are normal.

Survival of the sperm on extended testing is also a useful diagnostic test. The sperm survival test, or SST, is a method for testing the lifespan of the sperm. At 24 hours, sperm survival should be over 40% (i.e. 40% of the sperm sample should survive); conversely, lower survival rates correspond to lower pregnancy rates.

Additional testing for male factor infertility includes a physical exam, blood tests for FSH, prolactin, and testosterone, and an ultrasonography of the collecting tubes of the male reproductive system. In some cases, an assessment of DNA fragmentation can give an index of sperm quality as well.

One condition we encounter at our clinic is azoospermia, which is the absence of sperm in the ejaculate. This can occur from birth defects, injury or infection, or rare endocrine abnormalities. In azoospermia, a high FSH level indicates testicular failure. Insufficient levels of testicular hormones lead to an increase in the release of pituitary gland FSH to compensate. High levels of testicular hormones are often accompanied by testicular atrophy (small testicles). Testicular biopsy may confirm the clinical findings.

Men with testicular failure (and very low sperm counts) should be tested for Y-chromosome microdeletions and abnormal karyotypes, or chromosomal count. Microdeletions may be transmitted to offspring, resulting in fertility problems for boys born after treatment.

The most common abnormal karyotype is Klinefelter Syndrome, where the male has three or more sex chromosomes, instead of the normal two. Such chromosomal defects can have effects on children born after treatment, and men should receive genetic counseling and risk assessment prior to treatment. Men with testicular failure may still have partial sperm production. Detailed assessment with microscopic surgery may detect a sufficient amount of sperm to use with in vitro fertilization (IVF).

Obstruction is another type of male factor infertility, as potentially normal sperm cannot move from the testes to the ejaculate. Men with a normal FSH may have an obstruction in the vas deferens or any of the other collecting tubes that gather sperm from the testes. Men with congenital absence of the vas deferens (CBAVD) may be carriers of cystic fibrosis, and should be tested. Surgical obstruction, or vasectomy, is readily repaired. Microsurgical techniques, and an experienced surgeon, will increase success rates. The procedure may be attempted for many years after an initial vasectomy. More unusual obstructions can result after infection of the epididymis. Ejaculatory duct obstruction can be treated with a cystoscopic procedure. Obstructions can sometimes be repaired, but often a simple needle aspiration procedure (percutaneous epididymal sperm aspiration, PESA) will yield enough sperm to achieve fertilization with IVF.

The key treatment when working with low sperm numbers, whether in the ejaculate or obtained by needle aspiration or biopsy, is to perform in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). ICSI is when a highly trained embryologist uses micromanipulators to inject an individual sperm into an egg, optimizing for fertilization.

ICSI has become a common procedure, resulting in many pregnancies worldwide for men that otherwise could not have children. Sperm with a variety of abnormalities, ranging from low counts, to extremely low motilities, can be suitable for use. The DNA of the sperm is tightly compacted in ways that protect it from injury, even when the other components of the sperm do not function well. Injecting the sperm into the egg can bypass the barriers separating sperm and egg.

Another condition we encounter which can lead to abnormal sperm parameters is the presence of a varicocele. A varicocele is an enlarged vein along the upper part of the scrotum. The blood carried in these veins may elevate the scrotal temperature, and possibly carry toxic materials into the testicle, affecting sperm production. Only varicoceles that are palpable are thought to contribute to infertility. Ultrasound is sometimes used to confirm an uncertain diagnosis, but there is doubt whether subclinical varicoceles are associated with infertility. Varicoceles can be repaired, or various fertility treatments attempted, including sperm wash and insemination, and in vitro fertilization. The decision of treatment depends on both male and female factors, such as age, tubal disease, and ovulation disorders.

In closing, it is important to remember that infertility is not just a “female” issue and that men should engage in lifestyle habits that will not compromise their fertility. Furthermore, advancements in assisted reproductive technology (ART) have given men with infertility diagnoses newfound hope in their quest to build a healthy family.

– Philip Chenette, MD

ICSI Overview: Intracytoplasmic sperm injection (ICSI) is a technique used in the IVF laboratory to inject individual sperm into eggs. The procedure was developed in Belgium in the early 1990′s (Palermo et al., 1992) and it revolutionized the treatment of male factor infertility. Prior to ICSI, men with moderate and severe fertility issues had little or no chance of having their own genetic children. ICSI has so revolutionized the treatment of infertility that it is used in the majority (55.6%) of assisted reproductive technology cycles in the United States (CDC National Summary and Fertility Clinic Reports, 2003).

When IVF is performed without ICSI, it is common to incubate individual oocytes in a petri dish with about 100,000 sperm. Usually these sperm have been obtained by processing the semen in such a way as to be able to isolate sperm that look normal and swim energetically. Only the best 10% of the sperm in a normal semen sample are used, and in the petri dish, these compete for the honor of fertilizing the oocyte.

When ICSI is employed, individual sperm are isolated and forcibly injected into the oocyte by an embryologist. The oocytes have to be incubated in the enzyme hyaluronidase to remove the cumulus cells that surround them (naturally, these cells would be dislodged by the many sperm that try to penetrate the oocyte). Prior to injection, the sperm may be processed (as above) but often there are so few sperm available that processing is minimal. Once selected, the sperm is immobilized by breaking its tail. This is accomplished by dragging the injection needle across the tail until a visible kink or break can be seen. The immobilized sperm is then aspirated into the needle, which is pushed through the shell surrounding the oocyte and then through the cell membrane. The elasticity of the oocyte membrane is such that the embryologist must be rough with it to get through. Piercing the membrane is usually achieved either by poking it several times or by aspirating the membrane into the needle until it breaks. Once the membrane breaks, the sperm can be dropped inside the oocyte.

Technically, ICSI is one of the most difficult procedures to perform in the IVF laboratory and it requires a talented embryologist to do it well. As well as being responsible for choosing “the sperm”, the embryologist must work quickly and be firm enough to break the sperm tail and oocyte membrane while not being so aggressive as to kill the oocyte. ICSI has been so successful as a technique that it is now widely used in cases where there is no male factor infertility. In fact, of all the ICSI cases performed nationally in 2003, only 53% had a male issue (CDC, 2003). While ICSI is absolutely indicated for low sperm counts, decreased sperm motility, abnormal sperm morphology (size and shape) and surgically retrieved sperm, its use has expanded to include cases with anti-sperm antibodies, previous low fertilization with IVF, low oocyte numbers, frozen-thawed sperm and ejaculatory dysfunction such as retrograde ejaculation. In addition, ICSI is being widely used for patients having preimplantation genetic testing because it avoids DNA contamination during embryo biopsy by the many sperm that are usually attached to the shell of the embryo.

ICSI Risks: In assessing the risks of ICSI, we must first look at the procedure itself. In piercing the cell membrane, our greatest concern is in avoiding the area within the oocyte where the DNA is located. This is done by orientating the oocyte such that the polar body (a small packet of discarded DNA) is placed at the 12 or 6 o’clock position and the needle inserted at 3 o’clock. The polar body is the most practical indicator of where the oocyte DNA is located since it is created by the division of the oocyte’s total DNA just prior to ovulation. However, the DNA may not always be in the assumed place so a theoretical risk of damage exists, and chromosome breakage has been observed as being higher in ICSI-derived embryos when compared to conventional IVF embryos (Bergere et al., 1995; Edirisinghe et al., 1997).

In addition to DNA disruption or damage, the physical and biochemical disturbance that occurs could be significant. The injection procedure could introduce foreign material into the oocyte such as culture medium, seminal fluid with or without bacteria (Michelmann et al., 1998), viruses (Brossfield et al., 1999), or in theory, even prions (Lacey & Dealler, 1994) or foreign DNA.

Following the ICSI procedure, the fertilization process is known to be different than with conventional IVF with atypical decondensation of the sperm head resulting in delayed replication of the male genome. This is thought to result from the injection of the intact sperm into the oocyte since such sperm retain their acromosomal cap and perinuclear theca, both of which are normally lost as the sperm penetrates the shell of the oocyte. There is marginal evidence that the sperm sex chromosome is preferentially located in the anterior head and therefore might be impacted by the delayed decondensation caused by retention of the cap (Luetjens et al., 1999).

Currently there is no evidence that the miscarriage rate is different between ICSI and IVF pregnancies, and the incidence of prematurity and low birth weight babies (7.6% and 10.3% respectively for ICSI) is similar to that for IVF in large studies (Wisanto et al., 1995; Aytoz et al., 1998), but slightly higher than rates found in natural pregnancies. These outcomes have been confirmed in a large US-based study (Schieve et al., 2002) showing overall lower birth weight and higher perinatal mortality in children conceived with the help of reproductive technologies, but no significant differences between ICSI and IVF.

In the mid 1990′s ICSI had become a routine procedure in the world of assisted reproductive technology (ART) and was being widely used. However, reports surfaced indicating that the resulting children had a high incidence of chromosomal abnormalities (In ‘t Veld, 1995; Van Opstal et al., 1997). The immediate response from the ART community was a flurry of scientific papers refuting the findings, but ultimately the conclusions of the studies were confirmed by large scale, prospective systematic follow up studies on the ICSI children. Instrumental in these studies was the Brussels University where ICSI was invented. Thorough pre- and postnatal testing showed an abnormal karyotype in 2.6% of the ICSI pregnancies (Bonduelle et al., 1999) and in a subsequent study, 3% showed a chromosomal abnormality (Bonduelle et al., 2002). Novel chromosome abnormalities increased threefold (1.6% in ICSI vs. 0.5% in the general population) and these were mostly comprised of sex chromosome aneuploidies with a smaller number of autosomal structural anomalies. Inherited chromosomal abnormalities increased fourfold in ICSI pregnancies (1.4% compared to 0.3% in the general population) and this was related to the higher rate of existing chromosome abnormalities seen in the parents (mainly the fathers). It is important to point out that the incidence of these sex chromosome aneuploidies and structural abnormalities is inversely related to the number of sperm in the ejaculate and is therefore higher in ICSI fathers (4.8% vs. 0.5% in the general population), and interestingly also higher in ICSI mothers (1.5%: Van Assche et al., 1996). The structural chromosome abnormalities include deletions of sections of the Y chromosome in some men with low sperm counts which will be passed directly to sons created by ICSI.

We are fortunate that the children of ICSI are being widely followed and many solid studies have appeared and continue to appear on the incidence of congenital abnormalities (these are problems that cause impaired function and require medical or surgical intervention). The most common abnormality appears to be hypospadias (a urological condition where the urethra opens under the penis instead of at the tip, and which is correctable with minor surgery) which is increased in ICSI births (Wennerholm et al., 2000). However, when evaluating these cases, the increased risk for congenital abnormalities is often reduced or eliminated when confounding factors (maternal age, infertility, multiple pregnancy, familial and pregnancy history) are factored in (Ericson & Kallen, 2001). Nonetheless, it does appear as though ICSI and IVF children do have an increased odds ration (2.77 and 1.8 respectively) for malformations that need medical or surgical intervention in early life when compared to naturally conceived children (Bonduelle et al., 2005).

Concerns have also arisen about developmental delays in ICSI children as a result of a single paper (Bowen et al., 1998) that had them scoring lower on the Bayley Scales of Infant Development at 1 year of age when compared to IVF and naturally conceived infants. However, a good number of solid papers have since been published indicating that this finding is not holding up and that ICSI children are performing normally in psychological testing as well as in their cognitive and verbal skills using the Bayley and other scales of intelligence (Bonduelle et al., 1998; 2003 Ponjaert-Kristoffersen et al., 2004; 2005).

Finally, it is worth asking if gene expression is normal for ICSI children and are problems likely to arise as the children get older? In looking at gene defects, there is emerging evidence that ART children might be at a higher overall risk for genomic imprinting errors when compared to naturally conceived children. Genomic imprinting is a process that silences one gene from a parent, specifically so that the gene inherited from the other parent can do the work. The classic example is placental growth, which is controlled largely by paternal genes. Maternal genes for placental growth are deliberately inactivated since it is considered a conflict of interest for Mom’s genes to be involved in the regulation of how much of her resources the fetus gets. Problems arise when an imprinted gene is defective, because the perfectly good copy of the gene from the other parent has been switched off and therefore cannot work. Diseases such as Beckwith-Wiedmann and Angleman’s syndromes result from not having a functioning copy of a gene and preliminary evidence suggests that these might be more prevalent in IVF children (Gosden et al., 2003). Abnormal spermatogenesis is associated with an increase in defective genomic imprinting (Marques et al., 2003), but it is probably too early to tell if imprinting errors will occur more frequently in ICSI children. Angleman’s syndrome for example occurs at most at a rate of 1/200,000 IVF births, so the impact of ICSI will be difficult to measure. Similarly, retinoblastoma (a type of cancer of the eye that is caused by a genetic defect similar to what causes imprinted diseases) has been reported as slightly higher in IVF children (Moll et al., 2003) but further studies will be required to substantiate this observation and to ascertain the specific risk of ICSI.

ICSI is an aggressively invasive procedure that deposits a single sperm, usually from an infertile father, into the oocyte of a woman who has undergone fertility treatments. The specific risk of ICSI in offspring is an increased incidence of chromosomal abnormalities which may be caused by the procedure or by the parents, or both. ICSI is a routine and overly used procedure and patients should be educated as to the risks. Of the studies cited here, none of the children examined were older than 5 years. The long term hazards of the procedure, if any, remain to be determined. See below for the complete bibliography.

– Joe Conaghan, PhD

Bibliography:

Aytoz A, Camus M, Tournaye H, Bonduelle M, Van Steirteghem A, Devroey P. Outcome of pregnancies after intracytoplasmic sperm injection and the effect of sperm origin and quality on this outcome. Fertil Steril. 1998 Sep;70(3):500-5.

Bergere M, Selva J, Volante M, Dumont M, Hazout A, Olivennes F, Frydman R. Cytogenetic analysis of uncleaved oocytes after intracytoplasmic sperm injection. J Assist Reprod Genet. 1995 May;12(5):322-5.

Bonduelle M, Wilikens A, Buysse A, Van Assche E, Wisanto A, Devroey P, Van Steirteghem AC, Liebaers I. Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod. 1996 Dec;11 Suppl 4:131-55.

Bonduelle M, Aytoz A, Van Assche E, Devroey P, Liebaers I, Van Steirteghem A. Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum Reprod. 1998 Apr;13(4):781-2.

Bonduelle M, Camus M, De Vos A, Staessen C, Tournaye H, Van Assche E, Verheyen G, Devroey P, Liebaers I, Van Steirteghem A. Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum Reprod. 1999 Sep;14 Suppl 1:243-64.

Bonduelle M, Van Assche E, Joris H, Keymolen K, Devroey P, Van Steirteghem A, Liebaers I. Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Hum Reprod. 2002 Oct;17(10):2600-14.

Bonduelle M, Ponjaert I, Steirteghem AV, Derde MP, Devroey P, Liebaers I. Developmental outcome at 2 years of age for children born after ICSI compared with children born after IVF. Hum Reprod. 2003 Feb;18(2):342-50.

Bonduelle M, Wennerholm UB, Loft A, Tarlatzis BC, Peters C, Henriet S, Mau C, Victorin-Cederquist A, Van Steirteghem A, Balaska A, Emberson JR, Sutcliffe AG. A multi-centre cohort study of the physical health of 5-year-old children conceived after intracytoplasmic sperm injection, in vitro fertilization and natural conception. Hum Reprod. 2005 Feb;20(2):413-9.

Bowen JR, Gibson FL, Leslie GI, Saunders DM. Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet. 1998 May 23;351(9115):1529-34.

Brossfield JE, Chan PJ, Patton WC, King A. Tenacity of exogenous human papillomavirus DNA in sperm washing. J Assist Reprod Genet. 1999 Jul;16(6):325-8.

Centers for disease control and prevention. Assisted reproductive technology success rates. National summary and fertility clinic reports 2003 2005 Dec; United States Department of Health and Human Services.

Edirisinghe WR, Murch A, Junk S, Yovich JL. Cytogenetic abnormalities of unfertilized oocytes generated from in-vitro fertilization and intracytoplasmic sperm injection: a double-blind study. Hum Reprod. 1997 Dec;12(12):2784-91.

Ericson A, Kallen B. Congenital malformations in infants born after IVF: a population-based study. Hum Reprod. 2001 Mar;16(3):504-9.

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Human semen is a complex mixture of cells and fluids produced by the various components of the male reproductive system. The objective of sperm preparation is to remove the vigorously swimming sperm from this mixture, leaving behind the dead, dying or otherwise poorly swimming sperm, additional cells, enzymes and other factors that comprise the seminal fluid. A sperm cell is incapable of fertilizing an oocyte until it has separated from the seminal fluid.

We use a variety of separation techniques in the laboratory that are tailored to the procedure that the sperm will be used for, and modified according to the quality and type of sperm sample we receive. The average man manufactures about 250 million sperm in a 24 hour period. From a single ejaculate, we will only use 100,000 sperm for each oocyte that we have to inseminate in an IVF cycle. But for an intrauterine insemination, we want to get as many motile sperm as possible into the female reproductive tract, so we will therefore be using a much higher overall fraction of the sperm. Alternatively, for men who have no sperm in their ejaculate and for whom we have to retrieve sperm surgically from the testicle, we want to biopsy the minimum amount of tissue that will give us one sperm for every oocyte that has to be inseminated.

There are two general methods that we employ for the vast majority of sperm processing in the laboratory. The first is a density gradient centrifugation procedure in which the sperm sample is gently spun through 1-3 columns of a viscous solution of saline coated colloidal silica particles. The layers of silica are created by delicately layering different silica particle densities on top of each other in a test tube, and then layering neat semen on top. This method takes advantage of the fact that living sperm are dense compact cells that pass easily through the columns, while dead or dying sperm that are less dense due to leaky membranes are trapped with other cells and debris in the interfaces between the layers. The second method for preparing sperm takes advantage of the sperm’s natural swimming abilities by placing neat seminal fluid in proximity to some culture medium and allowing the sperm to swim from one to the other. There are many variations in this technique including the swim-up (semen is layered under the medium), or the converse method called the swim-down, and the actual method used depends mainly on the quality of the sperm sample. The swim-up is primarily used for samples that have good numbers of highly motile sperm from which only a small fraction needs to be recovered. The swim-down technique works better when sperm are swimming weakly and need the help of gravity to separate from the seminal fluid. For an individual with vanishing numbers of sperm (say a few hundred) we may use a swim-out technique. Here, the sperm are placed in the center of a small drop of medium and an embryologist will wait with a needle at the edge of the drop, picking up the first sperm to get there. One of the big criticisms of the ICSI procedure, where individual sperm are injected into oocytes, is that the embryologist chooses the sperm. However, with the use of the swim-out procedure, there is some degree of “natural selection” as we choose the sperm that are quickest in getting to the edge of the drop. We also choose sperm that are the normal size and shape, and that are free from defects (such as a bent neck) if we have the luxury to do so. In rare cases we have to use every sperm we have, so there’s no “selection” whatsoever. In most of the cases where we’re processing samples that have normal numbers of sperm, the sperm isolated by density centrifugation or by swim-up will be “washed” once or twice before being introduced to the oocytes. This involves suspending the sperm in a volume of culture medium and then centrifuging gently so that the sperm can be concentrated and removed from the medium, while leaving behind any trace of the silica particles or seminal fluid that may have carried over from the first processing step. Although sperm can be damaged by centrifugation, these steps are necessary to ensure that the sperm are free of contaminants that could prevent fertilization.

There are many other methods used to process sperm samples but we use them so rarely that they are scarcely worth mentioning. For example, samples with a high amount of debris can be filtered through glass wool or processed by sedimentation to clean them up before they undergo any of the procedures already described. In addition, we can treat a semen sample with chemicals in certain situations, but this again only happens under somewhat desperate circumstances. If a semen sample is extremely viscous or clotted, we can digest it using the enzymes amylase or chymotrypsin. If none of the sperm are moving we can treat them with pentoxifylline or caffeine to try to stimulate movement. When performing ICSI, we need to know that sperm are alive, and movement is our primary indicator. We can try to stimulate movement using drugs, but for the sperm that are to be used to fertilize the oocytes, we prefer to go drug-free. Here, we place the sperm into a hypo-osmotic solution (regular culture medium that has more water than normal) and as water enters living sperm their tails coil. These we can then inject into oocytes.

For patients that purchase frozen sperm from a sperm bank, the bank will usually offer the option of buying the sperm processed or unprocessed†. Processed sperm, usually labeled “IUI sperm”, costs a little more since the sperm bank has already prepared it for use. Unprocessed or “ICI sperm” is essentially neat semen that has been frozen. Women who do their own inseminations at home buy this type of sperm and inject it into their vagina after it is thawed. If you buy ICI sperm with the intention of having an intrauterine insemination, we will process the sperm as above to remove the seminal fluid and dead sperm. ICI sperm cannot be placed into the uterus since semen contains many contaminants such as bacteria, but also because semen can cause painful uterine contractions.

On a given day in our laboratory, one embryologist is primarily responsible for processing sperm samples, and each embryologist is assigned to this task about once a week. Each sample has different characteristics and the individual doing the processing must make informed decisions on the best approach for recovering the sperm that we need. It is an interesting and demanding area of the laboratory, but we enjoy the challenge of maximizing the potential of each sample that we receive.

– Joe Conaghan, PhD, HCLD

† For more information on frozen sperm and the products sold by sperm banks, see the “How do I Buy Sperm?” article in the April 2005 newsletter.

In graph A (pregnant) DNA fragmentation index is nice and low at 7.5%. You can see clearly that there are very few sperm (7.5%) with moderate or high fragmentation and that most of the sperm are bunched tightly together with very little fragmentation. These healthy sperm were able to establish and maintain a pregnancy.

In graph B (not pregnant), the sperm DNA is much more unstable and there is a fairly even spread of low, moderate and high fragmentation. The DNA fragmentation index is 65% and these sperm were unable to establish a viable pregnancy.

Intracytoplasmic sperm injection (ICSI), a procedure where a single sperm is injected into an egg, went into widespread use in the US in the early 1990′s. With it came the view that as long as a man had any sperm, he could father a child. In many ways ICSI was a remarkable procedure, allowing thousands of infertile males to have children. And ICSI worked even when the sperm didn’t swim well, had poor morphology or were surgically recovered from the epididymis or testicle. It appeared as though there was no physical obstacle to fertilization as long as a live sperm was available for injection.

Now, with over 10 years experience with this procedure, and regardless of sperm or egg quality, we understand that on average 70-80% of all eggs will fertilize following ICSI. If we physically place the sperm inside the egg, fertilization happens most of the time. However, fertilization is not a very reliable measure of sperm quality, or even egg quality, and the rate at which your eggs fertilize has little bearing on whether or not your embryos will implant after transfer. Eggs recovered from women aged 40 and older, where we know that egg quality is poor, will fertilize at the same rate as younger eggs. Similarly, sperm with poor morphology will fertilize eggs at the same rate as sperm with normal morphology.

After fertilization, if embryo quality is poor, or if embryos fail to implant after transfer, we tend to implicate the eggs as the likely source of the problem. It is very hard to pin the blame on the sperm and we usually have very little evidence that would implicate the male partner in the failure. After all, much time and effort was needed to get the eggs, the egg is mostly responsible for preimplantation development, and the developing embryo was placed safely in the uterus. The tiny sperm brought only the male’s genetic material or DNA, and we saw that that was safely inside the egg at fertilization.

Even when we start to worry about the DNA, eggs are much better known for genetic problems than sperm. Down syndrome is the classic example, as it is well known that the incidence increases with increasing maternal age. Genetic problems in children due to paternal age are less well known and in fact less than 10% of Down Syndrome cases arise as a result of a genetic error in the sperm.

In trying to visualize what DNA looks like, you have to think of a ladder. DNA is a double strand that is held together by the rungs, and the ladder is twisted and coiled. In sperm or eggs the DNA is organized on 23 distinct structures called chromosomes. Each chromosome is simply a very long twisted and coiled ladder.

When we count chromosomes in sperm and eggs, sperm have the right number about 90% of the time and for eggs this varies according to maternal age. For women over age 40, we would expect at least 50% of their eggs to have an incorrect number of chromosomes. These abnormalities don’t appear to stop eggs from fertilizing, but the majority of the resulting embryos either won’t implant or will miscarry early in pregnancy.

Because we know that sperm don’t carry a lot of chromosomal abnormalities, we have to dig deeper to find problems that may cause infertility. The sperm chromatin structure assay (SCSA) is a test developed to look at the integrity of the DNA. Basically it looks at the structure of the ladder and determines if the strands are coming apart due to broken rungs. The more severe the DNA fragmentation is, the less likely that the sperm can establish a viable pregnancy.

To have the test performed, we ship a frozen semen sample to Donald Evenson, PhD, in Brookings, South Dakota www.scsadiagnostics.com. There the sperm are assessed and any sample with less than 15% DNA fragmentation is considered normal. Levels of fragmentation up to 30% may cause reduced fertility, and men with greater than 30% fragmentation are considered to have significantly reduced potential to father a child.

Environmental stresses such as smoking, exposure to other chemicals or toxins, or any other chemical or physical stresses that the sperm may be subjected to may cause or contribute to high levels of sperm DNA fragmentation. In the testes it takes over 70 days to make each sperm, so the potential for exposure to stress is high. Consequently, it’s important for men to look after their health in the months leading up to their attempts to conceive. As always it’s good to eat well, exercise, avoid illnesses, hot tubs and exposure to toxins and take your vitamins. We particularly recommend vitamins C and E, beta-carotene and anti-oxidants for sperm health. We don’t routinely recommend the SCSA for our male patients since sperm fragmentation is likely to affect a very small number of men. The significance of a high fragmentation index is still under debate as there are reports in scientific literature of pregnancy successes despite a bad test result. Further, it is unclear what the prognosis is for men that succeed in reducing their fragmentation score by taking their vitamins and living healthier lives. An alternative solution for men with high fragmentation is to use donor sperm, however most couples choose to use their own sperm despite high fragmentation.

After my husband and I learned that we had no chance to become pregnant by natural means, we began to investigate IVF/TESE (sperm obtained by biopsy of the testes) with ICSI (Intracytoplasmic sperm injection) as a way to realize our dream of starting a family. We expected the procedures to be challenging to our bodies, minds, and finances.

We were also concerned about the frequency of twin and triplet births with IVF. As much as we hoped to have a child, we wanted to do everything we could to provide the best start for our child-to-be. We wanted to optimize our chances for a healthy full-term singleton pregnancy, natural childbirth, and breastfeeding, if we could become pregnant.

Dr. Carolyn Givens patiently answered our many questions about IVF and embryo cryopreservation and supported us when we made a choice that was quite unusual at the time: we requested that only one embryo be placed in my uterus during the IVF cycle and that any remaining embryos be frozen. I was 34 at the time and had never been pregnant.

Eight-cell embryo

We had the exceptional fortune that our first IVF/ICSI cycle in August of 1997 produced 13 beautiful embryos, and our transfer of a single fresh 3-day-old embryo during that cycle resulted in the birth of our son Benjamin nine months later.

I was still breastfeeding Ben in 2001 when we decided to try for a second pregnancy. Dr. Givens transferred a single 8-cell frozen embryo during an unmedicated natural cycle. We had explained to Ben that there was a little, little baby in Mommy’s tummy that we hoped might grow to be his brother or sister. About a week after the transfer, Ben said, “Mommy, the little, little baby in your tummy is crying.” A few days later, my period began, and I felt like crying too.

The next month, Dr. Givens transferred another frozen embryo, also without medication. Ben thought this embryo was happy, and he was right: she grew to be his sister Charlotte.

When we were considering the choice to have our embryos transferred one at a time, we were glad to learn that the expense of frozen embryo transfers was only a small fraction of that for the IVF/ICSI procedures. I found embryo transfers performed by Dr. Givens to be gentle and comfortable. Dr. Givens’ respect for our individual preferences made our infertility treatments a very positive experience. Our children have brought us unimaginable happiness.

– Camille, Redwood City

Most couples going through IVF or frozen embryo transfer choose to transfer at least two embryos in order to improve the chances of conception with any one embryo transfer procedure. As Camille’s story indicates, however, in younger patients with nice embryo quality and overall good chances for success, electing to transfer a single embryo is a viable option to avoid the risks of multiple gestation pregnancy. It also illustrates the benefits of embryo cryopreservation for having more than one child with a single IVF stimulation cycle.

– Carolyn Givens, MD