Clinical outcomes of microdissection testicular sperm extraction and intracytoplasmic sperm injection in Japanese men with Y chromosome microdeletions

Abstract Purpose We investigated the clinical results of Japanese men with Y chromosome microdeletions. Methods This study retrospectively examined 2163 azoospermic or severe oligozoospermic patients. We investigated the frequency of azoospermia factor (AZF) deletions and sperm retrieval rate (SRR) by microTESE in patients with these deletions, then analyzed the ICSI outcomes. Results Azoospermia factor deletions were found in 201 patients. SRR was significantly higher than that of the control group (74.0% vs 20.4%, P < .001). Thirty‐three couples underwent ICSI using testicular spermatozoa retrieved by microTESE, and eight couples underwent ICSI using ejaculatory spermatozoa. The fertilization rate and clinical pregnancy rate per embryo transfer cycle were significantly higher in the ejaculatory group than that of the testicular group (66.4% vs 43.7%, P < .001, 53.3% vs 24.7%, P = .03, respectively). When compared with the control group, the fertilization rate was significantly lower in the testicular group with AZFc microdeletions (43.7% vs 53.6%, P < .001). Conclusions Our study highlights that although microTESE in azoospermic men with AZFc microdeletions led to a higher SRR, ICSI outcomes of these men were worse than that of men without AZF deletions, even if testicular spermatozoa were retrieved.

Patients with azoospermia factor (AZF) deletions, especially AZFc microdeletions, have a chance to father offspring through sperm extraction techniques such as microTESE and in vitro fertilization with ICSI. [12][13][14][15] Stahl et al 16 reported that, using microTESE, sperm retrieval is higher from NOA patients with AZFc microdeletions than from idiopathic azoospermic men.
Azoospermia factor deletions are associated not only with disruption of spermatogenesis, but also with ICSI outcomes. Mateu et al 17 reported a high percentage of aneuploidies in spermatozoa and embryos from patients with Y chromosome microdeletions. In addition, both fertilization rate and embryo quality were significantly lower in patients with AZFc microdeletions, but pregnancy, implantation, and take-home baby rates were not significantly affected. 18 Although various findings have been reported, the phenotypic influence of AZF deletions clearly depends on the ethnic and geographic origin of the study population. 19 In the present study, we sought to clarify the frequency of each type of AZF deletion in Japanese azoospermic or very severe oligozoospermic patients, the SRR following microTESE in these patients with AZF deletions, and the ICSI outcomes from testicular or ejaculatory spermatozoa of these patients. Furthermore, we compared these results with their respective control groups.

| MATERIAL S AND ME THODS
A total of 2065 azoospermic and 98 very severe oligozoospermic patients were enrolled. All men suffering from infertility presented themselves to one of 16 reproductive centers in Japan from 2007 to 2017. They underwent semen analysis at least two times. After the diagnosis of azoospermia or very severe oligozoospermia (a sperm count of <1 million per mL), karyotype analysis and detection of Y chromosome microdeletions were performed on peripheral blood lymphocytes of these patients.

| MicroTESE procedure
If no spermatozoa were identified within the semen pellet, mi-croTESE was performed under local anesthesia by experienced surgeons as previously described. 20 We utilized the operating microscope and one transverse incision in the tunica albuginea through which spermatogenic tubules were selected for extraction. Almost all testicular spermatozoa retrieved by microTESE were cryopreserved for later ICSI use.

| Controlled ovarian stimulation and allocation
Most women underwent controlled ovarian stimulation with a gonadotropin-releasing hormone (GnRH) short antagonist or a GnRH short/long agonist protocol. 21,22 For ovarian stimulation, human menopausal gonadotropin was mainly used. Transvaginal ultrasound-guided oocyte retrieval was conducted 34-36 hours after the stimulation. One or two embryos were frozen by vitrification on the third day following oocyte retrieval, and the other embryos were placed in extended culture, from which blastocysts were frozen on day 5 or day 6.

| Endometrium preparation and embryo transfer
Hormone replacement therapy was employed for endometrium preparation in most cases. Briefly, oral ethinyl estradiol was administered from day 3 to attain an endometrial thickness ≥7-8 mm. At that time, patients were given luteal supplementation via intravaginal and/or oral administration of progesterone. Freeze-thawed ET (FET) of day 3 embryos or day 5 or day 6 blastocysts were scheduled based on the embryo and endometrium synchronization. Clinical pregnancy was established by ultrasonography at 5 or 6 weeks of gestation. Oral estradiol and progestin were continued until 10 weeks of gestation when pregnant.

| Statistical analysis
The data are presented as the mean ± standard deviation. Mann-Whitney U-test was used for continuous data and Fisher's exact test was used for categorical data to evaluate comparisons between the groups. A P value <.05 was regarded as significant.

| Incidence of AZF microdeletions in the Japanese population
One hundred and eighty-four AZF microdeletions were detected in the azoospermic patients, and 17 microdeletions in patients with very severe oligozoospermia. The number of patients found to have each AZF deletion is shown in Table 1. All men with AZFa, AZFb, AZFa + b, and AZFb + c microdeletions in Yq11 had azoospermia.
The 17 men with AZFc microdeletions had very severe oligozoospermia. The most common deleted region was AZFc (3.7%). Among the entire cohort, we performed subsequent examinations on cases in which the details of treatment were confirmed.
In contrast, we detected spermatozoa in only 20.4% (109/534) of the azoospermic men without AZF deletions. The characteristics and SRR of each group are shown in Table 2. The SRR was much higher in patients with AZFc microdeletions than that of patients without AZF deletions. Although three azoospermic men with AZFb + c microdeletions had also undergone microTESE following patient request, we did not detect spermatozoa.

| ICSI outcomes
Thirty-three couples underwent ICSI using testicular spermatozoa retrieved by microTESE, and eight couples underwent ICSI using ejaculatory spermatozoa. In this study, no couple underwent ICSI using both testicular and ejaculatory spermatozoa. The outcomes of ICSI are shown in Table 3. The fertilization rate was 47.6% (291/611).
We performed 92 embryo transfer (ET) cycles, and the mean number of replaced embryos was 1.42 per ET. The clinical pregnancy rate per ET cycle was 29.3% (27/92). The abortion rate was 22.2% (6/27). The fertilization rate was significantly higher in the ejaculatory group than in the testicular group (66.4% vs 43.7%, P < .001).
The clinical pregnancy rate per ET cycle was also significantly higher in the ejaculatory group (53.3% vs 24.7%, P < .03).
The two groups were also compared with their respective control groups. The fertilization rate was significantly lower in the testicular group than in the control group (218 ICSI treatments in 105 couples comprised of men without AZF deletions; 43.7% vs 53.6%, P < .001; Table 4). In contrast, there were no significant differences in any parameters of the ejaculatory group compared with the control group of 109 ICSI treatments in 46 couples consisting of men without AZF deletions (Table 5).  16 In the present study, we compared the ICSI outcomes between spermatozoa of different origins. Although the number of cases was limited, fertilization rate and clinical pregnancy rate were significantly higher in the ejaculatory group than in the testicular group. Furthermore, when compared with their respective control group, the fertilization rate was significantly lower in the testicular group, but there were no significant differences in In conclusion, NOA patients with AZFc microdeletions had a high percentage of successful sperm retrieval by microTESE, but the ICSI outcomes, especially fertilization rate, were not good. Although analysis of Y chromosome microdeletions is still not offered in some centers, our study emphasizes that diagnosis of Y chromosome microdeletions is critical for preconception genetic counseling and provides clinically valuable prognostic information to couples considering surgical sperm retrieval and ICSI.

ACK N OWLED G EM ENTS
We thank Okajima M, the president of LabCorp Japan Co., for detailed information about the Y chromosome microdeletion assay kit.