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To compare the frequency of Y-chromosome microdeletions in Japanese and African azoospermic …


Biology Articles » Reproductive Biology » Prospective assessment of Y-chromosome microdeletions and reproductive outcomes among infertile couples of Japanese and African origin » Materials and Methods

Materials and Methods
- Prospective assessment of Y-chromosome microdeletions and reproductive outcomes among infertile couples of Japanese and African origin

Between January 1998 and January 2003, male volunteers (n = 162) presenting for infertility evaluation and treatment at two centers were evaluated for Y-chromosome microdeletions via peripheral venipuncture. The study population consisted exclusively of Japanese (n = 113) and African (n = 49) males who, with their partners, sought infertility treatment either at St. Luke IVF Center (Japan) or Muhimbili National Hospital (Tanzania). Written informed consent was obtained from all study patients and the investigation was approved by the hospital's ethical committee. Subjects were partitioned into two groups based on sperm concentration: 1) nonobstructive azoospermia (NOA), and 2) oligozoospermia, defined as sperm concentration 6 ml.

The first group consisted of 47 males (26 Japanese and 21 Africans); the second group consisted of 115 males (87 Japanese and 28 Africans). None of the study patients were diagnosed with obstructive azoospermia. Six couples from the Muhimbili center were excluded from the study due to active STD. The GFX Genomic Blood DNA Purification Kit (Amersham Biosciences, Buckinghamshire, United Kingdom) was used to extract DNA from peripheral venipuncture samples as previously described [34]. Y-chromosome microdeletions were detected using a polymerase chain reaction (PCR) amplification with a specific sequence tag site (STS) using 24 sets of primers which allowed evaluation of the following sites: sY14, sY18, sY78, sY81, sY83, sY85, sY84, sY90, sY100, sY131, sY134, sY139, sY145, sY143, sY153, sY147, sY156, sY149, sY254, sY157, sY202, sY243, sY158, and sY159. Deletion of the loci was confirmed if the product of the expected size was not obtained after three single STS PCR experiments.

Four patients with NOA and Y-chromosome microdeletion underwent TESE. The samples were microscopically examined to search for sperm, which was cryopreserved as described previously [24]. TESE was successful in 2 of 4 azoospermic cases; after cryopreservation these couples subsequently underwent two ICSI cycles. The three Japanese oligozoospermic patients with Y-chromosome microdeletion produced fresh ejaculated sperm which was subsequently used for 6 ICSI cycles. Sperm morphology in each laboratory was examined by 2 observers [35]; classification of normal sperm morphology at our centers is: 14% = normal. No fathers or brothers of our male patients were available for testing. All Japanese couples who had a multifactoral diagnosis of oligozoospermia and tubal factor infertility were also assessed for Y-chromosome microdeletions and those found to have an intact Y-chromosome were assigned to the control group. Fourteen Japanese couples were initially considered for this group, but three were excluded because of endometriosis (n = 2) and leiomyoma (n = 1). These remaining couples (n = 11) constituted the control group and they underwent 19 ICSI cycles. Positive and negative controls were used for all AZF microdeletion tests. Thirteen fertile men with a sperm concentration of >20 × 106 /ml were used as positive controls, and twelve females served as negative controls.

Ovarian stimulation protocol, oocyte handling, laboratory procedures for insemination, measurement of sperm parameters, hormones, ICSI, and embryo and blastocyst grading were performed as previously described [35]. Using this protocol, only types I, II and III embryos were considered suitable for transfer and ≤ 3 embryos were transferred on day 3 after microinjection. For 6 cycles, embryos were cultured until day 5–6 and were transferred at the blastocyst stage. Clinical pregnancy was confirmed at 6 weeks via transvaginal ultrasound to establish embryonic cardiac activity.

Data were analyzed for equality of variance using the Levene's test. When p > 0.05 the variances were considered equal, a Student's t-test was performed, and p


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