Special Program of Assisted Reproduction

SPAR (Special Program of Assisted Reproduction) is a program offered to HIV discordant couples at the Bedford Research Foundation’s clinical laboratory [1]. The program takes advantage of ART (Assisted Reproduction Technology) procedures (including "sperm washing") to assist couples achieve a pregnancy who would otherwise risk transmitting the father's HIV infection to the mother and the child through intercourse.

SPAR employs extremely stringent safety standards; samples will only be submitted for "sperm washing" and cryopreservation that have first been screened for HIV virus particles and infected cells by specialized molecular biology tests. Only samples that have been determined to have an undetectable HIV viral burden will be submitted for infertility procedures.

Assisted Reproductive Technologies (ART), such as In Vitro Fertilization (IVF), treat many disorders of the female, but until recent years, treatment options for male factor infertility were limited, and donor sperm was routinely recommended to achieve a pregnancy. Advances in ART, however, have created new possibilities for men with male factor infertility, including those whose sperm counts have been decreased by cancer treatment and other diseases, and men with incurable, sexually transmissible virus diseases.

Infectious diseases transmissible by semen for which there is no cure include:

       - Hepatitis B (and possibly Hepatitis C)
       - Human T Cell Leukemia Virus (HTLV-1)
       - Human Immunodeficiency Virus (HIV)

History

SPAR began in 1994 at the New England Deaconess Hospital (now Beth Israel Deaconess Medical Center) as a support group for couples wishing to parent when one of them had an incurable sexually transmitted disease. The patient couples began raising money to fund the research needed to develop the methods to improve the safety of attempting pregnancy. By 1996 sufficient funds were available to begin, but a merger between the Beth Israel Hospital and the New England Deaconess Hospital (now Beth Israel Deaconess Medical Center) necessitated the creation of an independent Massachusetts public charity, the Assisted Reproduction Foundation (now the Bedford Research Foundation), to continue the work. Within two years, the methods for reducing, and perhaps eliminating, the risks of infection to the mother and the child were developed and Baby Ryan was born in 1999 (hear more about this at [2]).

The goal of SPAR was to provide semen testing by experts that would ensure improved safety of sperm that could then be shipped to infertility clinics near the couple's home. In this way, couples could be cared for in the same manner as other couples in their community. Fortunately, a few clinics were willing to help with this effort, and by 2002, seven collaborating clinics around the country were caring for couples living with HIV disease.

In the Spring of 2000 a ground breaking study was completed, giving significant insight to the role of semen producing organs in HIV anti-viral therapy.

HIV-1 in Semen: an isolated virus reservior: The Lancet

HIV Sperm Washing

In December, 1990, the First International Congress on AIDS and Reproduction was convened in Genoa, Italy. Dr. Ann Kiessling was invited faculty because she had published a Letter to the Editor in the journal, Fertility and Sterility, in 1989, entitled, "Retroviruses and Reproduction." At the Congress she was asked to speak about the role of Assisted Reproduction in assisting HIV infected men and women conceive.

Dr. Augusto Semprini, the founder of the "sperm washing" program in Milan, Italy, attended the congress. During the course of the congress, he met with Dr. Kiessling to discuss the options for helping HIV infected men with uninfected female partners parent. Dr. Kiessling's concerns at that time, and to the present, include the fact that HIV is present in semen in two forms: free virus particles and infected leukocytes. Free virus particles may be infectious, but they are more fragile than infected cells and can be "washed" away from the sperm. Although there are several laboratory methods for separating leukocytes from sperm which take advantage of differences in size, density and the fact that sperm are mobile, it is still possible that "washed sperm" could be contaminated with infected leukocytes which are not detected. It is important for couples going through assisted reproduction procedures to understand the limitations to laboratory assessment of sperm. Dr. Semprini decided to combine "sperm washing" with intrauterine insemination (IUI, described below). Dr. Kiessling decided to develop more reliable tests for HIV in semen before developing a program.

The biology of semen is complicated and not fully understood. Semen is a complex mixture of fluids and cells produced at ejaculation by organs, glands and tissues of the male reproductive tract (testis, epididymis, vas deferens, seminal vesicles, prostate and urethra). Sperm, on the order of 100 million per ejaculated specimen, are contributed by the testis, epididymis and vas deferens. Sperm mature and become motile following several weeks of development in the testis and epididymis. Some precursor cells which arrest part-way through development may also be ejaculated and are referred to as "non-sperm cells."

Seminal fluid and other cells are contributed by the seminal vesicles, prostate and urethra. All semen specimens contain leukocytes (white blood cells), but the number varies widely from specimen to specimen and they may arise from any of the semen producing organs. Semen leukocytes are also referred to as "non-sperm cells." Some precursor sperm cells look very similar to lymphocytes and differentiating the two cell types requires special techniques.

Sperm in the ejaculated semen specimen are counted either by a technician looking through a microscope, or by computer-assisted microscopy. For either method, a small portion (approximately one- tenth- of- a- drop) of a semen specimen, or a "washed sperm" specimen, is placed in a glass chamber with grid marks to indicate what percentage of the specimen is in the microcsope field at any one time. Thus, if one tenth of the small portion of the drop is being counted in the microscope field, then the number of cells or sperm in the entire tenth-of-a-drop is ten times the number of leukocytes and/or sperm seen in the microscope field. Therefore, the number of leukocytes and/or sperm in the entire semen specimen is thousands of times more than the number in the microscope field.

The most sensitive style of counting chamber is the one used to count blood cells (a hemocytometer); the limit of sensitivity is one leukocyte per grid counted which equals approximately one leukocyte in one tenth of a drop, or 10,000 cells per cc of semen (approximately 50 drops). Therefore, 9,999 leukocytes per cc of semen would not be detected in this chamber. The chambers used by computer-assisted microscopy are less sensitive, they hold approximately one one-hundredth of a drop (e.g. the Makkler Chamber). One leukocyte in one one-hundredth of a drop equals approximately 100,000 leukocytes per cc of semen. Therefore, 99,999 leukocytes per cc of semen (or "washed" sperm) would not be detected in the Makkler chamber.

The source of HIV infection in semen, either free virus or virus infected cells, remains unknown, despite many years of research in this area. What has become clear in recent years is that the burden of virus and infected cells varies widely from one specimen to the next and that the free virus particles present in semen are produced by a source of infection separate from the one that produces the infected cells. Therefore, more than one male reproductive tract organ is involved in the production of infected semen.

These considerations were important in designing the Special Program of Assisted Reproduction. In addition, in 1991, the Centers for Disease Control and Prevention in Atlanta published the opinion that intrauterine insemination with "washed" sperm from HIV infected men had not been proven to be safe.

For these reasons, it became clear that the safest method of assisted reproduction to help uninfected partners of HIV infected men conceive was to use sperm from semen specimens with an undetectable burden of virus in procedures of In Vitro Fertilization (IVF). In this way, the eggs would be fertilized in laboratory dishes with sperm washed from semen specimens that had either very low or no virus infection. The fertilized eggs could then be transferred to the woman's uterus as in standard IVF procedures. She would be exposed only to fertilized eggs, not sperm.

There were two ways to fertilize the eggs in the laboratory: either by exposure to 2 million "washed" sperm for one to two hours, or by injecting one selected sperm directly into an egg (intracytoplasmic sperm injection, ICSI). Moreover, because sperm can be cryopreserved (frozen to stay alive) for long periods of time, it would be possible for couples to receive care by fertility clinics close to their homes because frozen sperm from tested semen specimens could be shipped to those clinics. By shipping only sperm from specimens with an undetectable burden of virus, exposure of laboratory personnel, medical staff and other patients would be greatly reduced .

Therefore, the Special Program of Assisted Reproduction (SPAR) was designed with the following goals:

1. The development of a sensitive, reliable procedure for testing semen specimens for HIV particles and HIV-infected cells 2. The establishment of a program of In Vitro Fertilization for couples with infectious disease infertility 3. The demonstration that uninfected partners of HIV infected men could achieve a pregnancy without themselves or the baby becoming infected 4. The development of methods for shipping semen specimens to the testing laboratory for sperm cryopreservation so couples could be cared for by infertility clinics close to their homes

External links

• Bedford Research Foundation Clinical Laboratory
• Bedford Research Foundation