New Leaders In Fertility & Endocrinology

Infertility is a prevalent condition with significant societal and psychological impact. Thirty percent of couples seeking treatment for infertility are unable to conceive solely due to male related problems. Impaired sperm performance in conjunction with other female factors is noted in an addditional 20 percent of patients.

Normal male fertility requires completion of a daunting sequence of elaborate processes to include spermatogenesis, sperm transport, accessory gland function, along with timely sperm deposition through adequate coitus. Prior to acquiring fertilization ability the sperm undergoes complex morphological and biochemical changes. This process of capacitation allows hyperactivated motion characteristics, zona pellucida binding, acrosome reaction and oocyte penetration. Once within the oocyte, decondensation is necessary for male pronuclear formation; thus completing the most essential goal of spermatozoa: delivery of genetic material to the oocyte. Natural reproduction offers other obstacles requiring adequate sperm function. After vaginal deposition, a fraction of the total ejaculated sperm enters the cervical mucus, potentially extending viability of the male gametes to four days. The fertilizing spermatozoa must traverse the female reproductive tract to reach the distal fallopian tube, the usual site of fertilization. In the absence of patent fallopian tubes or surgically correctable tubal obstruction, couples have to resort to In Vitro Fertilization (IVF) for procreating. During IVF successful gamete interaction becomes dependant upon oocyte quality, sperm count, motility, morphology and ability to capacitate followed by acrosome loss (1,2).

Brijinder S. Minhas, Ph.D., HCLD, MBA
Barry A. Ripps, MD, FACOG, ACGE.
Global Infertility Solutions
Kenilworth, Illinois
New Leaders In Reproductive Endocrinolgy and Infertility
Pensacola, Florida

Genetics of Male Factor Infertility

Spermatogenesis is the complete process of germ cell development from mitotic division of spermatogonia to the meiotic division of spermatocytes and differentiation of spermatids. The genetics of this process encompasses the genetic program expressed by the germ cell, as well the somatic components of the testis. The successful production of a sufficient number of functional spermatozoa is dependent on the fidelity of gene expression in both germ cells and somatic cells during spermatogenis. Mutations in genes responsible for the intricate process may result in germ cell dysgenesis or dysfunction, ultimately causing infertility(3).

Recent estimates from several studies agree that unto 30% of male factor infertility cases may have a genetic basis. Most of these patients have severe oligoasthenoteratozoospermia or azoospermia. Currently genetic testing focuses on three primary modalities: karyotypic analysis for abnormalities in chromosome number or structure, Y-chromosome microdeletion screening and mutational assessment of the cystic fibrosis transmembrane conductance regulator gene.

Chromosomal aberrations are numerical or structural and may be detected by routine karyotyping. These aberrations can be manifested as Ring chromosomes, Reciprocal translocations, Balanced translocations, Pericentric inversions or Robertsonian translocations and are more prevalent in infertile men with potential consequences for future children.

The most common abnormality of chromosome number is Klinefelter's syndrome (47,XXY), a condition affecting 1 in 500 men and up to 15% of all azoospermic men. The classically described phenotypic manifestations (tall stature, feminine hair distribution, etc) are frequently absent in these men. Klinefelter's mosaics (47,XXY,46XY) may have some sperm in the ejaculate; nearly all men with the true condition are azoospermic. Biologic paternity in azoospermic men with the nonmosaic Klinefelter's can only be achieved through testicular sperm retrieval (TSTE) and ICSI(4,5).

The Y-chromosome conceptually can be divided into two major areas based upon the presence of repetitive copy or single copy DNA sequences. The area corresponding to the cytogenetic nomenclature designation q12 is the heterochromatic or fluorescent portion of the Y-chromosome is composed of repetitive copy DNS which is characterized by multiple copies of pentameric tandem repeats (TTCAA). The remainder of the Y-chromosome from Yq12 to the Yp telomere is the euchromatic region consisting of single copy or low copy repeat sequences.

Twenty five years ago Tiepolo and Zuffardi analyzed the karyotypes of over 1000 infertile men and noted that in a small percentage of the patients a partial deletion of the distal long arm of the Y-chromosome (Yq). On further investigation they noted that that the men missing this portion of the Y-chromosome were azoospermic. They postulated the existence of an azoospermic factor (AZF) on the Yq that contained genes necessary for spermatogenesis(6). Modern techniques in molecular biology have allowed for the development of detailed Y-chromosome maps and several microdeletions in the Y-chromosome have been identified which were too small to be discerned by standard karyotyping.

Deleted segments of the Y- chromosome do not occur randomly but rather seem to involve specific, nonoverlapping regions that are known as AZFa, AZFb, AZFc and AZFd. It has been postulated that multi-repeat sequences surrounding these regions predispose them to breakage or deletion. The size and location of Y- chromosome deletions is extremely important. Although ultra short deletions have been observed in normal fertile, substantial deletions have only been detected in men with severely compromised spermatogenesis.

AZFc deletions have been transmitted to male offspring both by natural conception and through IVF-ICSI. It is thought that these offspring will have impaired fertility however there are any other serious health consequences to Y micro-deletions. There is evidence that Y-chromosome deletions may lengthen as it is passed down to the next generation.

Brandell et al., have recently reported that AZFb deletions either alone or in combination with another AZF region uniformly predicts the absence of mature sperm when contemplating testicular sperm extraction.

Cystic fibrosis (CF) is a common fatal autonomic recessive disorder with an incidence of 1:2000 and a carrier frequency of 1:20. The CF gene is located on chromosome 7 and encodes for a cAMP-regualted chloride channel found in secretary epithelial cells. This particular gene, the cystic fibrosis transmembrane conductance regulator (CFTR) and 800 different mutations have been identified. In addition to the life threatening pulmonary and pancreatic manifestations of CF, these patients almost invariably have bilateral congenital absence of the vas deferens (CBAVD) and azoospermia. Using CFTR mutation analysis it has recently been shown that 80% of men with CBAVD are found to have aberrations in the CF gene and suffer from sub-clinical CF symptoms including mild elevations of sweat chloride concentrations and chronic sinusitis. The vast majority of men with CBAVD have normal spermatogenesis. The wide range of mutations and their different degree of penetrance make genetic counseling and risk estimation for offspring difficult.

If CF mutations are detected in both partners, the risk for their children having CF or CBAVD clearly increases. However if the female partner tests negative, the chance of having a son with CBAVD is less than 1%. Extreme caution should be exercised before proceeding with ART in patients with CBAVD.

Semen Analysis

A comprehensive semen analysis remains a cornerstone in the routine infertility analysis. Semen quality is conventionally determined according to the concentration, motility and morphology of the spermatozoa in the ejaculate. Human spermatozoa show a great variation in size and structure compared to other mammals. Normal fertile ejaculates contain spermatozoa exhibiting considerable variations not only in size and shape of the head but also in the degree of nuclear vacuolation, persisting cytoplasmic droplets, mid-piece disturbances and tail abnormalities.. Of all semen parameters sperm morphology has best correlated with fertilizing capability. Additional parameters of semen viscosity, liquefaction and spermatozoa longevity are also considered in an ART evaluation. With the discovery of ICSI and its clinical application worldwide, zero motility (even tail twitching) is the only parameter that causes concern to the practitioner.

Intracytoplasmic Sperm Injection

Microsurgical fertilization techniques namely Zona Drilling, Partial Zona Dissection followed by Subzonal Sperm Insertion met with limited clinical success. In 1992 an aggressive Subzonal attempt in Belgium resulted in deposition of the sperm directly into the cytoplasm and ICSI was born(7). After the first report appeared in Lancet, ICSI technology spread quickly and revolutionized the treatment of male factor infertility. Prior to the advent of ICSI, male factor infertility cases during ART therapy had the poorest clinical outcomes. Couples with severely compromised semen parameters were not accepted into IVF programs of the ones that were accepted the couples failed to proceed to embryo transfer as the initial hurdle of achieving fertilization many times was not crossed.

Most couples with severe male factor infertility can now be treated utilizing ICSI, which requires only one sperm cell with a functional genome and centrosome for the fertilization of each oocyte. Spermatozoa can be surgically retrieved from the epididymis or testis in the case of absence ejaculatory sperm. Artificial insemination with donor sperm is currently only used when ICSI with spermatozoa from the ejaculate, epididymis or the testis cannot be used or has failed. It also may be used for couples that prefer artificial insemination because of financial, psychological, ethical or genetic considerations.

In established ICSI programs routinely 75% of injected oocytes fertilize, displaying two pronuclei and two polar bodies 16 hours after injection. Normally 75% of the zygotes cleave and develop into embryos of sufficient quality warranting embryo transfer. Clinical pregnancy rates are in the 50% range with delivery rates near the 30% mark. It is hard to compare pregnancy rates as different numbers of embryos are transferred in programs worldwide. A more rigorous indication of the quality of a program can be deciphered by comparing embryo implantation rates, live take home baby rates and a low multiple gestation rate. Reducing the numbers of multiples is a key focus of ART practitioners globally. The associated risks to mother and children in multiple gestations, plus the astronomical health care costs both immediate and long term, in cases of obstetrical complications or developmental disabilities, have captured center stage attention.

ICSI bypasses many of the upstream events which prepare the sperm for egg penetration. During the procedure a selected immobilized spermatozoon is injected directly into the cytoplasm of a human oocyte after removal of cumulus cells and corona radiata, with particular attention to the rupture of the oolemma. If the oolemma is not ruptured completely allowing deposition of sperm within the cytoplasm, poor fertilization results are obtained. The selection of the sperm cell for injection should invove attention to motility (distinguishes viable from dead), normal appearing morphology and maturity.

Normal fertilization and pregnancy rates can be obtained with the use of elongating, elongated or mature spermatids. Fertilization with round spermatids and spermatocytes has been far less successful and is controversial. Recent reports (8,9), however indicate that fertilization rates can be improved when injecting round spermatids or their isolated nuclei provided that oocyte activation is facilitated by the use of a calcium ionophore (A23187).

Reports on follow-up studies on children born from ART therapies are limited in number and scope. The studies are flawed and it is hard to get a clear understanding of the associated risks. Two recent studies (10,11) in Lancet highlight the need for case controlled studies taking into account various confounding variables. The Australian study compared the medical and developmental outcome at one year of 89 ICSI children with 84 IVF and 80 naturally conceived children. Using the Bayley Scales for Infant Development they found no significant difference in the incidence of major congenital malformations or major health problems in the first year of life. However the Mental Development Index was significantly lower for ICSI children (especially boys). Professor Van Steirteghem's in the same issue reported no differences in mental development when 201 ICSI and 131 non-ICSI IVF children at 2 years of age were compared in reference to the general population. Further studies are clearly warranted and appropriate scenarios need to be developed for patient counseling.

Summary

We conclude this chapter with a brief description of three clinical cases where the merits of ICSI utilization are crisp:

Case I

Thirty nine year old presented in 1994 for her second baby. Her husband had a sperm count of 100,000 per ml. In the previous cycle the couple had used donor sperm in 1992.

With ICSI on board in 1994, the patient was stimulated using standard down regulation and gonadotropins producing 12 oocytes. Eight oocytes were mature and injected resulting in 6 zygotes. Four embryos were transferred on day 3 resulting in the delivery of a baby girl. This couple have a child from donor sperm- IVF and another from husband sperm-ICSI.

Case 2

Thirty year old presented with severe tubal disease. Husband had normal semen parameters. In the first IVF cycle l3 oocytes were retrieved. The oocytes were inseminated with 50,000 motile cells each. The next morning it was determined that all the oocytes were unfertilized and there was hardly any binding of sperm to the zona pellucida. The couple returned for a second attempt with ICSI. After stimulation 6 oocytes were retrieved and all deemed mature and injected resulting in 6 zygotes. Four embryos were transferred on day 3 resulting in the delivery of twins.

Case 3

Forty two year old had failed IVF three times at another center. In each attempt she had produced a lot of eggs with good fertilization but poor embryo quality. We decided to perform ICSI in hopes that inseminating sperm waste products were contributing to poor embryo quality. We transferred four good quality embryos which resulted in the delivery of a singleton.

Should ICSI Replace Conventional Insemination?

References

1. Minhas, BS: Efficiency enhancement of manipulated male and female gametes. Semin Reprod Endocrinol. 12:177-183,1994.

2. Minhas, BS, and Ripps, BA: Methods for enhancement of sperm function. Frontiers in Bioscience. 1: 65- 71,1996.

3. Mak, V, and Jarvi, KA: The genetics of male infertility. Journal of Urology. 156:1245-57,1996.

4. Palermo, GD, Schlegal, PN, Hariprashad, JJ,et al.: Fertilization and pregnancy outcome with lntracytoplasmic sperm injection for azoospermic men. Hum Reprod. 14(3):741- ,1999.

5. Palermo,GD, Schlegel,PN, Sills, ES, et al.: Births after lntracytoplasmic sperm injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter's syndrome. N Engl J Med. 338(9): 588- ,1998.

6. Tiepolo, L , and Zuffardi, O: Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet. 34:119-124.

7. Palermo, GD, Joris, H, Deroey, P, Steirteghem, AC: Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 340:17-18,1992.

8. Tesarik, J, Mendoza C, Greco,E: In vitro culture facilitates the selection of healthy spermatids for assisted conception. Fertil Steril. 72: 809-813,1999.

9. Tesarik, J, Mendoza C, Greco, E: The activity(calcium oscillator?) responsible for human oocyte activation after injection with round spermatids is associated with sperm nuclei. Fertil Steril. 74:12451247,2000.

10. Bowen,JR, Gibson, FL, Leslie, GI,Saunders DM: Medical and developmental outcome at I year for children conceived by intracytoplasmic sperm injection. Lancet. 351:1529-1534,1998.

11. Bonduelle, M, Joris H, Hofmans, K, Liebaers,I, Steirtegehem, AC: Mental development of 201 ICSI children at 2 years of age. Lancet. 351:1553, 1998.