Fred Gebhart

A familiar laboratory assay is emerging as a reliable indicator of the likelihood of success in assisted reproductive techniques (ART). Researchers from the United States and Egypt have established a cutoff value for sperm DNA damage based on a simple comet (single cell gel electrophoresis) assay.  

"We can use comet values to predict which couples have a good chance of getting pregnant," Hussein Abdelrazik, MD, TS (ABB), andrologist and supervising clinical embryologist in Cairo, and consultant at the Center for Reproductive Medicine, Cleveland Clinic, told 

 at the American Society for Reproductive Medicine annual meeting. "There is a significant correlation between comet assay results and the likelihood of pregnancy using ART." 

The comet assay classifies sperm DNA damage in a given semen sample into mild, moderate, and severe strata, Dr. Abdelrazik said. Sperm showing 28% or greater severe DNA damage are not likely to result in pregnancy for couples undergoing intracytoplasmic sperm injection (ICSI). Sperm with less than 28% severe DNA damage are likely to produce pregnancy. 

The comet assay is commonly used to assess DNA damage to single cells in research and clinical settings. The technique was first described in 1984, and is named for the appearance of damaged DNA that is displaced during electrophoresis from the cell like a comet's streaming tail. Both tail length and tail moment, the fraction of cellular DNA that has been fragmented and pulled into the tail during electrophoresis, are used to assess DNA damage. 

Dr. Abdelrazik said that the comet assay correlates well with both TUNEL and assays of DNA fragmentation, but comet is simpler and easier to perform. Until now, there have been no data to show a cutoff value for sperm DNA damage and pregnancy outcomes in any form of ART using the comet assay. 

Investigators analyzed sperm samples from 48 couples who requested in vitro fertilization. The project was a collaboration between Kasr El-eni Hospital-Cairo University, the Nile Badwri Hospital in Cairo, and Cleveland Clinic. All of the couples had used ICSI in an attempt to achieve pregnancy. 

Semen samples were given conventional and computerized analyses. A portion of each sample was then subjected to comet assay. Samples were divided into two fractions for duplicate analysis and 100 sperm cells from each fraction, or 200 cells from each man, were examined and scored for DNA fragmentation. 

Sperm DNA fragmentation was classified as mild, moderate, or severe. The comet score is based on tail moment, which reflects the portion of sperm DNA that has been damaged and has migrated from within the cell during electrophoresis. A tail moment of greater than 40 was considered to indicate severe sperm DNA fragmentation. 

Couples whose ICSI resulted in pregnancy had a higher percentage of mild sperm DNA fragmentation (

=.023) and a lower percentage of severe sperm DNA fragmentation (

=.01) than did couples who did not achieve pregnancy. There was no predictive value for moderate sperm DNA fragmentation. 

Among couples with less than 28% severe sperm DNA damage, 51.6% of the women became pregnant, while none of the women in the couples with 28% or more severe sperm DNA damage became pregnant. The 28% cutoff (

<.017) showed 100% sensitivity and 53.1% specificity, Dr. Abdelrazik reported. 

Additional data are needed to confirm the results, he said, but comet appears to provide a reliable, affordable, easy-to-perform predictor for pregnancy in ICSI. 

"If the score is 27% or lower, the chances of getting pregnant are good," he concluded. "If the score is 28% or above, the chances of pregnancy are not good at all." 

Articles

There is a significant correlation between Comet assay results and the likelihood of pregnancy using ART.

Comet assay may predict assisted reproductive techniques pregnancy success

The likelihood of successful sperm extraction may be higher at later-stage sperm maturation arrest (MA), according to a study conducted by researchers at Baylor College of Medicine, Houston, and presented at the American Society for Reproductive Medicine annual meeting. 

Sperm MA is widely recognized as a contributing factor to male infertility, but there is little information in the literature to help characterize its stages. Researchers in Japan have reported that men with earlier stages of MA have higher levels of follicle stimulating hormone (FSH). Recent data from Cornell University showed that men with MA who had normal FSH levels had a lower sperm retrieval rate, lower fertilization rate, and lower pregnancy rate compared to other men with nonobstructive azoospermia.  

The ways in which MA itself might affect the success of mature sperm extraction remain largely unknown. The Baylor researchers hypothesized that patients with earlier stages of MA might be associated with higher levels of FSH resulting from increased sertoli cell or other testicular damage. They also suggested that the likelihood of successful sperm extraction is higher at later-stage MA. 

Dr. Weedin and his co-researchers retrospectively reviewed all patients who underwent site-specific sextant biopsies (testis mapping) by a single urologist at Baylor between 2002 and 2008. They identified 149 patients with nonobstructive azoospermia and 32 patients with severe oligospermia (sperm density less than 10,000 per mL). 

Patients were grouped by their major and any minor histopathology patterns. Categories included sertoli-cell only (SCO), early maturation arrest (EMA), late maturation arrest (LMA), and hypospermatogenesis (HS). EMA was defined as maturation arrest up to the level of the primary spermatocyte and LMA from the secondary spermatocyte to the formation of the immature spermatid. 

Each of the patient groups was analyzed for serum FSH concentration, testicular volume, chromosomal abnormalities (including karyotype abnormalities and Y-chromosome microdeletions), and the presence of sperm during testicular mapping. 

The analysis found 61 patients with MA and 20 with concomitant SCO histology. The mean serum FSH level in men with EMA was 18.7 compared to 7.6 mIU/mL in men with LMA, a statistically significant difference (

=.0043). FSH levels correlated positively with testis pathology, but not with the probability of obtaining sperm at biopsy. The frequency of successful sperm extraction during testis mapping increased with the later stages of MA, from 14.3% in EMA to 26.7% in SCO/mixed MA, 37.5% in mixed MA, and 46.1% in LMA. 

"Men with earlier maturation arrest have higher FSH levels, which matched the findings of one other report," Dr. Weedin said. "There is also a higher chance of finding sperm in men with later stages of maturation arrest than in the earlier stages. Some men with late MA potentially have full maturation somewhere in their testicle that we might be finding, or not. There is a difference between the early MA group and later MA that could have some clinical correlation in the future." 

Researchers identified a potential genetic component in more than three-quarters of the men in the review. Karyotypic abnormalities were seen in 15.5% of men, but there was no correlation with stage of maturation arrest. Microdeletions in the Y chromosome were far more common and varied by MA classification: 36.8% in EMA, 15.6% in LMA, 23.5% in mixed MA, and 2.1% in all other patients (

"Some men seem to have a gene that is deleted or nonfunctioning that prevents their sperm from maturing," Dr. Weedin said. "At some point, we may be able to manipulate the genetic pathway to alleviate the problem." 

The likelihood of successful sperm extraction may be higher at later stage sperm maturation arrest (MA), according to a study conducted by researchers at Baylor College of Medicine, Houston that was presented at the American Society for Reproductive Medicine annual meeting.

Sperm maturation arrest: Better late than early

Injecting an immunofluorescent dye into the testes may eventually help clinicians locate individual sperm and improve the yield from microdissection testicular sperm extraction (mTESE), researchers from the University of Iowa, Iowa City, reported at the American Society for Reproductive Medicine annual meeting. 

But it's too early to suggest immunofluorescence-assisted mTESE for infertile patients. All of the experiments to date were performed in mice-and current protocols include removing entire testicles for microdissection. 

About half of the six million U.S. couples who are infertile have at least one component of male infertility. Thirty percent have pure male factor infertility, Dr. Greenhalgh said. 

mTESE has been used successfully in cases of nonobstructive azoospermia, but there can be significant difficulty in visually identifying areas that contain sperm within the testes. mTESE has a 40% to 50% sperm retrieval rate, which makes any technique that can increase sperm retrieval rate a potential improvement over current practice. 

"We hoped that this immunofluorescence technique would help improve the procedure," Dr. Greenhalgh said. 

The idea is that tagging individual sperm with fluorescent markers could help clinicians identify foci of sperm during mTESE. Identifying sperm foci could boost sperm retrieval for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). 

Preliminary in vitro testing using a fluorescent dye (fluorescein isothiocyanate, or FITC) conjugated with mouse anti-Human acrosomal IgM antibody (HS-14) produced easily visible sperm. The dye bound readily with both the head and tail of previously extracted mouse sperm, producing bright, glowing sperm that were easy to identify.  

A group of 13 fertile mice and eight sterile mice that had been genetically engineered for early spermatogenic arrest were anesthetized and the testes exposed. Researchers used a microsurgical knife to make a small incision near the tunica albuginea near the vascular pedicle to expose the seminiferous tubules. HS-14 plus a small amount of crystal violet powder was injected into the exposed seminiferous tubules. After the dye was injected, the testicles were removed for microscopic examination. 

Sperm were easily visible in all 13 of the fertile mice, Dr. Greenhalgh reported. Fluorescent dye bound to both the head and the tail of sperm in 22 of the 26 testes. It was particularly easy to visualize fluorescing motile sperm, he added, but even immobile sperm were readily visible. No sperm were seen in any of the 16 testes from the sterile mice. Histopathologic review of the sterile mice testes also failed to show the presence of any sperm. 

Whether immunofluorescent-labeled sperm are viable for IVF or ICSI is still unknown, Dr. Greenhalgh added. There are no sperm-specific toxicity data on HS-14 or the antibody binding process itself and no data on the effects of binding or unbinding the fluorescent label on sperm viability, motility, and other properties. There are also no data on potential effects of immunofluorescent labeling on sperm DNA or a resulting embryo. 

"Immunofluorescence may be the next step for improving sperm retrieval rates in microdissection mTESE," he said, "but it isn't as easy as it looks. There are some major engineering and biological questions to be addressed." 

Injecting an immunofluorescent dye into the testes may eventually help clinicians locate individual sperm and improve the yield from microdissection testicular sperm extraction (mTESE), researchers from the University of Iowa, Iowa City, reported at the American Society for Reproductive Medicine annual meeting.

Immunofluorescence may improve microdissection testicular sperm extraction outcomes

Men who respond quickly to varicocele repair have a better chance of fathering children than men who take longer to respond do. That's the clinical pearl from an early study of infertile men at the Medical College of Wisconsin, Milwaukee, reported at the American Society for Reproductive Medicine annual meeting. 

"This finding is based on clinical experience," Anand Shridharani, MD, a urology resident working with Jay Sandlow, MD, told 

"What we saw in practice was that men whose semen parameters improve earlier achieve pregnancy more frequently than those who improve later or do not improve at all. We crunched the numbers to prove this finding." 

Surgical varicocelectomy improves semen parameters in between 60% and 70% of men, yielding pregnancy rates of up to 50%. However, few data identify prognostic factors for successful pregnancy following varix ligation. 

Drs. Shridharani and Sandlow conducted a retrospective study of 144 men with clinically palpable varicoceles who underwent microsurgical subinguinal varicocele repair between September  2003 and November 2007. Their goal was to compare pregnancy rates among couples in which the man showed improvement in at least one semen parameter within 3 months of surgery with those couples in which the man took longer to respond. 

"In the couples where the man improved at 3 months, about 70% got pregnant," Dr. Shridharani said. "The number of couples who got pregnant was lower for men who improved at 6 months or longer after surgery. The bottom line is that if your semen parameters improve at 3 months, you are much more likely to get pregnant." 

Men in the study had a semen analysis before surgery and at 3-month intervals following surgical repair of the varicocele. Of the 144 couples represented in the retrospective chart review, 117 couples had semen analysis data, 70 couples had pregnancy data, and 56 couples had both semen analysis and pregnancy data. 

Semen parameters measured included a 50% or greater increase in concentration, motility, or total progressively motile/ejaculate as measured at 3 months compared to 6 months or later. Pregnancy could be achieved by natural means or by intrauterine insemination (IUI), but not by in vitro fertilization. 

Overall, semen parameters improved in 62% (73/117) of men and 49% of couples (34/70) achieved pregnancy by natural means or IUI within 12 months following surgery. But among couples who became pregnant, 84% of men showed improvement in semen parameters at 3 months compared with 10% of those with improved semen parameters at 6 months or later and 6% who showed no improvement in semen parameters 12 months postoperatively. 

Among couples in which the man showed improved semen parameters at 3 months, 68% became pregnant. Among couples who did not become pregnant, 50% had improved semen parameters at 3 months, 8% showed improvement at 6 months, and 44% showed no improvement in semen parameters at 12 months after varicocele repair. 

"If semen parameters have not improved at 6 months, we can suggest that couples might want to look seriously at assisted reproductive technologies, especially in the setting of advanced maternal age," Dr. Shridharani said. 

"We hope this study offers information that clinicians can use in preoperative and postoperative counseling. If these results are reproducible in larger numbers of patients, we may make more inferences based on the numbers. Ultimately, offering this information to patients will allow them to make better decisions about seeking further treatment after varicocele repair and to improve their allocation of resources." 

Men who respond quickly to varicocele repair have a better chance of fathering children than men who take longer to respond do.

Author | Fred Gebhart

Articles

Comet assay may predict assisted reproductive techniques pregnancy success

Sperm maturation arrest: Better late than early

Immunofluorescence may improve microdissection testicular sperm extraction outcomes

Varicocelectomy response rate: Pregnancy predictor