New Sperm Shaker Set To Improve IVF Success Rates

Scientists have developed a ground-breaking method for testing the quality of a sperm before it is used in IVF and increase the chances of conception.

Researchers at the University of Edinburgh, funded by the EPSRC (Engineering and Physical Sciences Research Council), have created a way of chemically 'fingerprinting' individual sperm to give an indication of quality. Scientists can then consider whether the sperm is healthy enough to be used to fertilise an egg as part of an IVF treatment.

The sperm are captured in two highly focussed beams of laser light. Trapped in what are essentially 'optical tweezers', an individual sperm's DNA properties are identified by the pattern of the vibrations they emit in a process known as Raman spectroscopy. This is the first time this process has been used to evaluate DNA damage in sperm.

Dr Alistair Elfick, lead scientist on the project, said: "In natural conception the fittest and healthiest sperm are positively selected by the arduous journey they make to the egg. What our technology does is to replace natural selection with a DNA based 'quality score'. But this is not about designer babies. We can only tell if the sperm is strong and healthy not if it will produce a baby with blue eyes."

In the past quality tests of sperm have mostly been carried out on the basis of shape and activity. While these do give some indication of health of the sperm they do not give its DNA status.

There are established tests for sperm DNA quality but they work by cutting the cells in half and tagging them with fluorescent dye a process that kills the sperm and renders it useless. This new process does not destroy the sperm, so if it is found to have good DNA quality, it can still be used in IVF treatment.

Conception rates in both IVF treatment and intercourse are at around one in four. By selecting the best quality sperm it is hoped this new process could both increase a couple's chances of conception and give the child the best potential start in life.

The research is currently in a pre-clinical phase, and if successful could be available to patients in the next five to ten years.

Increased Fertility Problems For Men Over 40

Scientists have found further evidence to suggest that, like women, fertility drops in men as they age, heard delegates at the annual meeting of the European Society of Human Reproduction and Embryology. The research, led by Dr Stephanie Belloc of the Eylau Centre for Assisted Reproduction in Paris, France, said that the results - the first to show such a strong paternal effect on pregnancy and miscarriage rates - will have important implications for couples wanting to start a family.
'I think it's important to consider not only the woman, but both members of the couple in natural conception but also in assisted reproduction', Belloc told the Times, adding: 'We believe that the use of IVF should be suggested to infertile patients where either party is over 35 years of age'.
The researchers recorded rates of pregnancy, miscarriage and birth in 12,000 couples undergoing fertility treatment in the form of intrauterine inseminations (IUI), where sperm is injected into the woman's uterus while she is ovulating. They also examined the quality and quantity of the sperm, including their ability to swim, size and shape.
The results showed that, independent of the woman's age, the chances of miscarriage rose from 16.7 per cent if the man was 30-35 years old, to 32.5 per cent if he was over 40. Although the impact of the female 'biological clock' on fertility has been widely studied, this is the first time that such a strong paternal effect on reproductive outcome has been shown, said Belloc. 'Some recent studies have established a relationship between the results of IUI and DNA damage, which is also correlated with is also correlated with a man's age, suggesting that it might be an important factor, but until now there was no clinical proof', she said.

Folic acid improves a man's chance of becoming a father

US scientists have found a possible link between low dietary folate levels and abnormal sperm in men. The findings, from the University of California, Berkley, and the Lawrence Berkley National Laboratory, provide further evidence that healthy diets aid fertility. Folate is a soluble B vitamin found naturally in foods such as citrus fruits, green leafy vegetables and pulses. The synthetic form, folic acid, can be taken in dietary supplements. The benefits for women of high folate levels are well established in preventing birth defects, but this is the first time a reproductive benefit for men has been shown. The normal
recommended daily allowance (RDA) of folate for humans is 200 micrograms, and 400 micrograms for women trying to conceive and during pregnancy. The study, published in the journal Human Reproduction, looked at samples from 89 healthy, non-smoking men aged between 22 and 80, and information about their diet and supplementary intake. They found that men who ingested high levels of folate - between 722 and 1150 micrograms per day - had 20-30
per cent lower levels of abnormal sperm than men with low folate intake. The researchers were looking at forms of 'aneuploidy' in the sperm. Aneuploidy is an abnormal number of chromosomes in cells. If these abnormal sperm fertilise an egg, the resulting fetus can have the wrong number of chromosomes, something that can lead to chromosomal disorders such as Down's syndrome. Other risk factors also include miscarriage during pregnancy. The researchers targeted specific chromosomes - chromosomes 21, X and Y - as these are associated with the common types of aneuploidy in live births.
Brenda Eskenazi, professor of epidemiology and maternal and child health at UC Berkeley's School of Public Health, was the co-principal investigator of the study. She says: 'in previous studies, we and others have shown that paternal micronutrient intake may contribute to successful conceptions by improving the quality of the sperm. This study is the first to suggest that
paternal diet may play a role after conception in the development of healthy offspring'. The scientists warn that this is not conclusive evidence yet, and both men and women should take a serious look at their diet and lifestyle when trying to conceive. Smoking, drinking excessively and unbalanced diets are highly likely to affect fertility. In the US, folic acid has been added to breads, flour, cereals and other grain products since 1998 to ensure women get their RDA of folate. A
decision on whether folic acid should be added to bread and flour in the UK is due next year.

'Miracle baby' conceived from 22-year-old sperm

A Canadian couple successfully conceived a baby boy after using sperm that had been stored 22 years, two months and two weeks - a likely record for Canadian fertility treatment. The longest-known storage period for sperm resulting in a live birth is 28 years, according to a 2005 data report in the American journal Fertility and Sterility. Jacek was born in Langley, Canada on 1 November 2007 thanks to Mike Kuzminski's discovery that a Calgary clinic, where he had stored sperm years ago prior to cancer treatment which rendered him infertile, had continued to store his sperm despite no contact or payment from Kuzminski in 22 years.Kuzminski, now a 43-year-old, was diagnosed with Hodgkin's lymphoma at 18. Because chemo and radiotherapy treatment caused 20 per cent of patients to become infertile in the 1980's - a risk that is significantly lower today - his doctor recommended that Kuzminski freeze his sperm. He did and then underwent three years of on-and-off treatment that led to his later-confirmed infertility. When he and his wife Kristin married in 2003, they had accepted that they would not be able to have children. Kuzminski had since forgotten he had frozen sperm before his treatment years ago until his sister reminded him. 'I had assumed that after 15 or 20 years the hospital had gotten rid of my sperm', he said but instead surprisingly found that the clinic had kept it and he owed Rs. 90,000 in storage fees.
Kuzminski seems to have benefited from Canada's current policy indecision regarding gamete storage time-limit guidelines for abandoned frozen sperm. He would not have been so lucky in the UK, where gametes are stored for a maximum of ten years, now with the option to request a five year extension.
Despite great anxiety regarding use of a limited supply of sperm and low success rates of fertility treatments, Jacek was born after undergoing only two intra-uterine insemination (IUI) procedures. They have enough sperm remaining for 19 more attempts but are focusing on their new parenthood and postponing consideration of whether they will try to have another child.

Sperm damage 'passed to children'

Sperm defects caused by exposure to environmental toxins can be passed down the generations, research suggests. Scientists say fathers who smoke and drink should be aware they are potentially not just damaging themselves, but also their heirs. Tests on rats showed sperm damage caused by exposure to garden chemicals remained up to four generations later. The US study was presented to the American Association for the Advancement of Science (AAAS). It suggests that a father's health plays a greater role in the health of future generations than has been thought. A team from the University of Idaho in Moscow tested the effects of a hormone-disrupting fungicide chemical called vinclozolin on embryonic rats. The chemical altered genes in the sperm, including a number associated with human prostate cancer. Rats exposed to it show signs of damage and overgrowth of the prostate, infertility and kidney problems.The defects were also present in animals four generations on. The scientists admitted that the rats were exposed to very high levels of vinclozolin.

But they argued that their work shows that once toxins cause defects in sperm they can be passed down the generations. Professor Cynthia Daniels, from Rutgers University in New Jersey, said men who drank a lot of alcohol had been shown to have increased rates of sperm defects; and nicotine from tobacco found its way into seminal fluid as well as blood.Professor Daniels said: "We need to open up our eyes and look at the evidence. "My advice to young couples would be moderation. Substances that have an impact on reproduction are often also carcinogenic. "If I was a young man I would not drink very heavily and not smoke two packets of cigarettes a day while I was trying to conceive a child."

Professor Neil McClure, a fertility expert at Queen's University Belfast, UK, said the DNA in sperm cells was more tightly packed than in other cells, and so, to some extent, was protected from damage. However, once sperm cell DNA was damaged, it had no mechanism by which to effect repairs. He said: "There is no doubt that if you smoke like a chimney or drink vast amounts of alcohol it will result in sperm damage, and probably damage in the DNA of the sperm. "My advice to any man trying for a baby would be to lead as healthy a lifestyle as possible."

Less Dud, More Stud Sperms

The University of Michigan's sperm sorter consists of a penny-size silicon chip divided into two channels. Semen is dripped onto one side, a saline solution on the other. Where the channels meet, the healthy, or motile, sperm swim over to the saline channel, leaving the dead or slow sperm behind. The healthy sperm are then collected for in vitro fertilization. University of Michigan scientists have developed a new technique to sort out the swimmers from the duds in semen, which could lead to a more efficient way for men who suffer from low sperm counts to make babies.

Current methods use centrifugation, which spins the sperm at very high speed. But the technique isn't efficient because live sperm are pelted with dead ones, causing a significant number of viable sperm to die in the separation. "We can harvest motile sperm from samples where there is a low number without causing any damage to the sperm," said team member Dr. Gary Smith, associate professor of obstetrics and gynecology at the University of Michigan Health System.

In men with healthy sperm counts, each ejaculation contains about 200 million to 400 million sperm. For these men, in vitro fertilization using centrifugal separation is relatively easy because it only takes 10 million to 20 million motile sperm to fertilize an egg in a petri dish. But men with low sperm counts have had less success. "For men with very low sperm count, these other techniques are often unable to recover any motile sperm," said Shuichi Takayama, assistant professor in the Biomedical Engineering program at the University of Michigan, where the new sorter is being developed. Other methods to pinpoint live sperm in a low sperm sample require a microscope and a lot of patience. The new sperm sorter may put an end to this laborious hand sorting. "It may mean that instead of having a well-trained technician, you could have one that is not so well-trained," said Dr. Mark Surrey, of the Southern California Reproductive Center in Beverly Hills.

So far the sorter has fared well in trials. In one test the team used a sample in which only 44 percent of the sperm was motile. After going through the sorter, the count of motile sperm went up to 98 percent. The sorter employs microfluidics, a branch of physics and biotechnology that examines the microscopic flow of fluids. Within the device everything is pushed downstream by gravity and surface tension. The Michigan team notes, however, that more tests are needed before the sorter becomes a mainstay in doctors' offices. Smith said he expects it will be ready for clinical use in one to two years.

Sperm Penetration Assays (SPA, "Hamster Tests")

There have been many attempts made to develop a Laboratory test that will accurately predict the ability of a human sperm to fertilize a human egg. Dr. Aitken and his group many years ago demonstrated a correlation between sperm movement characteristics and sperm fertilizing ability as evaluated by the zona pellucida-free hamster egg penetration test. In this test, the species specific barrier to penetration (not fertilization) is removed from the ova (eggs) of the hamster. These oocytes are then exposed to prepared sperm from the man being tested. One of the most widely used accessory tests in the evaluation of advanced sperm function, the fusion between human sperm and the hamster oocyte (egg) is nearly identical to that occuring with the human egg. Fusion with the vitelline membrane of the oocyte is normally initiated by the healthy sperm's plasma membrane that lies over a special section (equatorial segment) of the sperm head in a sperm that has prepared itself for normal fertilization. This test relies upon the ability of sperm that are tested in the laboratory to undergo the necessary reactions to fertilize an egg. Because the sperm prepare themselves in a slightly different manner in the laboratory than in the body, false negatives (fail in the hamster test but normally fertilize the partner's human egg) have been reported. This test may be used as a screen to asist in determining which men may need the assistance of "ICSI" fertilization. There is some feeling that if a man's sperm are able to penetrate the hamster eggs in the laboratory, there is a higher likelihood that his sperm will ultimately be able to fertilize a human egg if so exposed. This test is not uniformly accepted, due to the high false negative (no penetration of the hamster egg, but wife gets pregnant anyway) rate and the sometimes seen false positive (penetrates the hamster egg but does not fertilize human eggs in vitro) rate of this test. Global experience has been that good performance in the hamster test can provide some limited reassurance of the likelihood that a man's sperm will be able to achieve fertilization if given the chance. If men fail the hamster test, most centers rely upon in vitro fertilization with ICSI. This protocol has provides excellent success rates in men whose sperm function remains questionable. It should be noted that most men that fail the hamster test, are able to achieve normal fertilization with ICSI.

Computer Assisted Semen Analysis (CASA)

The use of computer asisted semen analysis has advanced the ability to study and understand sperm function as it relates to human infertility. The major advances have been in the ability to more accurately determine sperm concentration (counts) and motility (movement). Generally, sperm are "looked" at by a computerized digitizing tablet through a microscope. The computer has been "taught" by the laboratory personnel what sperm look like, and how they move. When the computer then "sees" a sperm under the microscope, it is able to draw a digitized picture of each individual sperm, including the speed and path this sperm takes while moving under the microscope. A great deal has been learned about the normal and abnormal "micro"characteristics of sperm employing this method. The method is, however, not foolproof. The computer is only as intelligent as it's programmer. Small changes in the computer program can alter the sperm calculations significantly. The computers must constantly be monitored and updated. In most laboratories, all grossly abnormal CASA assays are always verified by both a repeat analysis as well as with a "hands on" human second look opinion. We feel that any abnormal sperm count must be verified by a manual counting and assesment method.

Sperms & The Laboratory

Laboratories performing sperm "counts", in general, vary in the details that they provide the physician requesting the "count". A general sperm count as part of a fertility evaluation should include the total density or count (20 million per ml or above), and the motile density (8 million per ml or higher). The motile density is perhaps the most important part of the semen analysis, as it reports the total number of sperm thought capable of progressing from the site of sperm deposition to the site of fertilization. This value is essential in both allowing a determination regarding whether or not a semen analysis is "normal", as well as in providing prognostic information should advanced reproductive medical assistance be required. (Numbers in italics are what "normal" values should be.)

Definitions of "abnormal" counts:
• Polyzoospermia: Excessively high sperm concentration.
• Oligozoospermia: Sperm count less than 20 million/ml
• Hypospermia: Semen volume < 1.5 ml
• Hyperspermia: Semen volume > 5.5 ml
• Aspermia: No semen volume
• Pyospermia: Leukocytes (germ fighter cells) present in semen
• Hematospermia: Red blood cells present in semen
• Asthenozoospermia: Sperm motility < 40%
• Teratozoospermia: > 40% of sperm seen are of abnormal form
• Necrozoospermia: Nonviable ("dead") sperm
• Oligoasthenozoospermia: Motile density < 8 million sperm/ml

Sperm Morphology (Shape and Appearance)
The evaluation of sperm size, shape and appearance characteristics should be assesed by carefully observing a stained sperm sample under the microscope. The addition of colored "dyes" (stains) to the sperm allow the observer to distinguish important normal landmarks (characteristics) as well as abnormal findings. Several methods of staining sperm are used, and the method employed should be one with which the examiner is comfortable and experienced.

Several different shapes or forms of human sperm have been identified and characterized. These forms fall into one of four main categories: normal forms, abnormal head, abnormal tail and immature germ cells (IGC).
Normal forms
Normal sperm have oval head shapes, an intact central or "mid" section, and an uncoiled, single tail.

Abnormal heads
Many different sperm head abnormalities may be seen. Large heads (macrocephalic), small heads (microcephalic) and an absence of identifiable head are all seen in evaluations. Tapering sperm heads, pyriform heads (teardrop shape) and duplicate or double heads have been seen. Overall (gross) abnormalities in appearance may be termed "amorphous" changes.

Abnormal tails
Coiling and bending of the tail are sometimes seen. Broken tails of less than half normal length should be categorized abnormal. Double, triple and quadruple tails are seen and are abnormal. Cytoplasmic droplets along the tail may indicate an immature sperm.

Immature germ cells (IGC's)
White blood cells (WBC's, germ fighters) in the semen should rarely be seen. It is very difficult to distinguish between an immature germ cell and a WBC. Because the presence of WBC's in the semen (pyospermia) can be a serious concern, if a report of "many IGC's" is delivered, it becomes very important to assure that these cells are not, instead, WBC's.

Sperm "Motility" (Movement)
Sperm motility studies identify the number of motile (moving) sperm seen in an ejaculate specimen. Here again, as in many other sperm studies, many laboratories use "normal" values that are out of date and inaccurate. Many labs will assess sperm motility upon receipt of the specimen, and again at hourly time intervals for four to twenty four hours. It is well known that sperm motility is a temperature dependent sperm function, so the handling and processing of specimens is critical. It is for this reason that we, except in very rare instances, require that specimens be evaluated only in a laboratory such as our own, where we are able to tightly control laboratory conditions. We have found the repeated testing of sperm over time for motility adds little to the evaluation of motility over the initial sperm motility assessment. Sperm are known not to survive well for extended periods of time in semen, and in nature, sperm very rapidly leave the semen to enter the cervical mucus. Many laboratories consider "normal" sperm motility to be 60% or greater. Our own studies, in agreement with many others have found men with 50% or greater sperm motility to be "normal".

Asthenozoospermia
Decreased sperm motility. If found to be present, exam should be repeated to assure that laboratory conditions did not cause the problem. Frequent causes: abnormal spermatogenesis (sperm manufacture), epididymal sperm maturation problems, transport abnormalities, varicocele. These conditions should all be looked for if sperm motility is repeatedly "low".

Necrozoospermia
A total absence of moving sperm. It is vital, if sperm are seen, but are not moving, to carry out studies (vital stains) to see if the sperm seen are alive. It is possible to have sperm with normal reproductive genetics that are deficient in one or several of the factors necessary to produce motility. We have achieved several successful pregnancies employing microinjection of healthy, non motile sperm directly into the egg (ICSI).

Chemical and Biochemical Semen Characteristics
Semen acid-base balance (pH)
The pH of semen is measured using a specially treated paper blot that changes color according to the pH of the specimen that it is exposed to. The pH of normal semen is slightly alkaline ranging from 7.2 to 7.8. Prostatic secretions are acidic while the secretions of the seminal vesicles are alkaline. Therefore, alterations in pH may reflect a dysfunction of one or both of these accessory glands. The pH of semen has not been generally found to have a major influence on a man's fertility potential.

Color and Turbidity
Semen is normally translucent or whitish-gray opalescent in color. Blood found in semen (hematospermia) can color the semen pink to bright red to brownish red. The presence of blood in semen is abnormal and should be reported. The presence of particles, nonliquified streaks of mucus or debris requires further evaluation.

Liquefaction
Semen is normally produced as a coagulum. The specimen will ususally liquify within 30 minutes. The failure to liquify within one hour is abnormal. Excellent methods for correcting this problem in the laboratory are available.

Viscosity
Nonliquefaction and excessive viscosity are two separate conditions. Viscosity is measured after complete liquefaction has occured. Viscosity is considered "normal" if the liquefied specimen can be poured from a graduated beaker drop by drop with no attaching agglutinum between drops. The role of hyper (excessive) viscosity is being studied, but it seems possible that htis condition may interfere with the ability of sperm to travel from the site of deposition into the cervix or uterus.