Improved sperm-washing method removes small risk of assisted reproduction for serodiscordant couples

Japanese scientists have improved upon current sperm-washing methods in order to assist HIV-positive men and their HIV-negative female partners to more safely conceive children. By measuring single copies of HIV DNA and proviral DNA and modifying the current swim-up method of isolating sperm from semen, they have eliminated the risk of the HIV-negative mother or unborn child being infected with HIV, according to data published in the April 24^th issue of the journal, AIDS.

Sperm washing is an experimental option for couples who wish to conceive where the male is HIV-positive and the woman HIV-negative. This technique was pioneered in Milan and so far the Italian programme has undertaken more than two thousand inseminations and several hundred IVF cycles. So far, although none of the women has seroconverted and all of the children born have tested HIV-negative, there still remains a very small theoretical risk to the woman of HIV transmission.

This risk occurs because a study by Zhang and others has shown that HIV may be present as proviral DNA in seminal cells even when HIV RNA is undetectable in the blood. It has also not been determined whether HIV attaches to sperm, or whether sperm can be infected with HIV, since studies so far have been inconclusive.

Scientists in Tokyo first developed a very sensitive HIV RNA/DNA test that was capable of measuring single copies of HIV by selecting and improving techniques in three main steps. First they collected HIV virions and infected cells via a centrifuge. They then extracted viral RNA and DNA with silica gel membrane technology. Finally they detected viral RNA and DNA by nested PCR. At each step, there was zero apparent loss in recovery, resulting in a protocol that can detect a single virion or infected cell even in the presence of 1.8 million sperm per sample.

They then revised the swim-up method of collecting HIV-free sperm that had been developed in Milan. This improved method cuts the test tube in half, and prevents the sperm suspension at the top of the test tube from possibly being infected by the sediment at the bottom of the test tube. The sperm suspension is then introduced into the bottom of another test tube and the sperm that swim up to the top of the tube are then recovered.

The scientists report on the first 52 HIV-positive men who participated in a clinical study along with their HIV-negative female partners. A total of 39 men were on potent anti-HIV therapy. Twenty-three of the men had a plasma viral load of less than 50 copies/ml; the remaining thirteen had a median viral load of 1500 (range 54-31,000) copies/ml. The other sixteen untreated men had a median viral load of 17,500 (range 70-100,000) copies/ml.

Four men out of the 52 in the study had no detectible sperm: two on treatment with an undetectable viral, one on treatment with a detectable viral load, and one not on treatment.

Among the 48 remaining participants, the median sperm count of the men with an undetectable viral load on therapy was 35 million per ml; for those men on therapy with a detectable viral load, the median sperm count was 41 million per ml; and for the men not on therapy the median sperm count was 47 million per ml.

Following the swim-up method, there were a median of 1.5 million (range 0-11 million) sperm/ml amongst the 48 men, and the motility rate was 100%. The investigators were able to collect 73 semen samples from the 48 men, and no HIV RNA or proviral DNA was detected in any sperm suspensions collected after the swim-up procedure.

A total of 31 couples underwent intracytoplasmic sperm injection (ICSI, where a single sperm is injected through the outer wall of the egg) and twelve couples underwent in vitro fertilisation (IVF, where many sperm are introduced to a woman's egg). Again, neither HIV RNA nor proviral DNA was detected in the fertilised egg prior to the embryo being transferred to the womb.

Of the 43 female partners, 20 conceived and 27 babies were born. Neither the women nor the babies tested positive for HIV antibodies, HIV RNA or proviral DNA.

The authors say that they have "demonstrated that it is possible to collect spermatozoa with evidence of the absence of HIV-1 RNA and proviral DNA from semen of HIV-infected males."

They conclude by emphasising that "whatever method is used for assisted reproductive technique and for removal of HIV from semen to reduce the risk of secondary transmission, it is essential to confirm the absence of HIV-1 RNA and proviral DNA in the sperm preparation used for the assisted reproductive technique with the most sensitive test possible."