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Four layer discontinuous gradient for HIV
  1. K Kakimoto,
  2. Y Ando,
  3. A Yoshioka
  1. International Medical Centre of Japan, Tokyo, Japan
  1. Correspondence to:
 K Kakimoto;

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Artificial insemination using processed semen is a risk reduction option, if they want children, for serodiscordant couples in whom the man is HIV positive. The main aim of this study was to develop a single semen processing technique to reduce HIV transmission risks to HIV negative wives without infection and to obtain better quality sperm.


After ethics committee approval and written informed consent, normozoospermic semen was provided by two asymptomatic HIV carriers. Discontinuous four layer density gradient, whose fractions (Fr) were 1.065 (Fr 4), 1.085 (Fr 3), 1.110 (Fr 2), and 1.135 (Fr 1), was prepared with Puresperm. Semen washed with Hank’s solution was laid on this gradient and centrifuged at 400 g for 30 minutes. The specimen of each fraction was extracted to determine sperm quality and to detect HIV RNA and proviral DNA using RT-PCR and PCR, respectively. Lymphocytes of an HIV non-carrier were co-cultured for 4 weeks with each fraction. HIV p24 antigen and proviral DNA after co-cultivation with each fraction were determined by indirect immunofluorescence assay and polymerase chain reaction (PCR), respectively.


The percentage collection of sperm from Fr 1, Fr 2, Fr 3, and Fr 4 was 3% (SD 2%), 32% (9%), 19% (8%), and 10% (4%), respectively. Motility rate was 55% (19%), 94% (4%), 57% (25%), and 19% (11%), respectively. HIV proviral DNA and HIV RNA were detected only from Fr 4. HIV p24 antigen was observed in the lymphocytes co-cultivated with Fr 4 and from the positive control, but was not observed in other fractions. HIV proviral DNA was not detected from Fr 2 or Fr 3 (tables 1 and 2).

Table 1

Sperm characteristics and detection of HIV in each fraction

Table 2

Detection of HIV p24 antigen and proviral DNA after 4 weeks’ co-cultivation with each fraction and carrier’s PBL


HIV discordant couples have a risk of transmission generally if they wish to have a baby.1,2 Semprini et al3 reported continuous gradient centrifugation followed by a swim up procedure, and Marina et al4 carried out a similar method but HIV was detected in 5.6% of 107 samples. However, the condition of the sperm, after these processes, was not always sufficient for intrauterine insemination.

We have developed a novel semen single processing technique to reduce HIV RNA and HIV proviral DNA to undetectable levels in the fraction whose sperm quality was higher than others. Furthermore, this fraction was confirmed to have no HIV infectivity in vitro. This method appears to be an attractive alternative for HIV discordant couples.


KK and YA contributed to laboratory work; AY referred HIV positive volunteers.