R J M E Romanian Journal of Morphology & Embryology

Transcription

1 Rom J Morphol Embryol 2016, 57(2 Suppl): CASE REPORT R J M E Romanian Journal of Morphology & Embryology Pregnancy resulting from IMSI after testicular biopsy in a patient with obstructive azoospermia CONSTANTIN-CRISTIAN VĂDUVA 1), CARMEN CONSTANTINESCU 2), MIHAELA MIRELA RADU 3), ALICE ROXANA VĂDUVA 4), ANDREI PĂNUŞ 5), MIHAELA ŢENOVICI 6), DAMIAN DIŢESCU 7), DINU FLORIN ALBU 8) 1) Department of Obstetrics and Gynecology, University of Medicine and Pharmacy of Craiova, Romania 2) Department of Obstetrics and Gynecology, Railroads Clinical Hospital, Craiova, Romania 3) Department of Embryology, HitMed Medical Center, Craiova, Romania 4) Department of Endocrinology, HitMed Medical Center, Craiova, Romania 5) Department of Urology, Emergency County Hospital, Craiova, Romania 6) Department of Pathology, Railroads Clinical Hospital, Craiova, Romania 7) Department of Obstetrics and Gynecology, Constantin Brâncuşi University, Târgu Jiu, Romania 8) Department of Obstetrics and Gynecology, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Abstract In this article, we report a case of pregnancy obtained in an infertile couple diagnosed with severe male factor infertility. The couple attended for fertility examination reporting a history of 10 years of infertility. The cause of infertility was obstructive azoospermia. The treatment consists of in vitro fertilization (IVF). The ovarian stimulation of female patient was done with antagonist protocol and after ovarian puncture was obtained nine oocytes. The urologist performed testicular sperm extraction (TESE). There were selected nine sperm cells by intracytoplasmic morphologically selected sperm injection (IMSI). For this purpose, we used an inverted microscope with high magnification equipped with 60 air objectives with modulation contrast illumination. After intracytoplasmic sperm injection (ICSI) of sperm into the oocytes there were obtained six normal embryos from which three embryos were transferred into the uterus. A singleton pregnancy was achieved which was completed with birth of a healthy baby in time. This successful outcome shows that use of IMSI and ICSI procedures are really useful in selection of best spermatozoa obtained by TESE in treatment of obstructive azoospermia. Keywords: azoospermia, in vitro fertilization, oocytes, testicular sperm extraction. Introduction Azoospermia, defined as complete absence of sperm from the ejaculation, is present in less than 1% of all men and in 10 to 15% of infertile men. Although there are many causes of azoospermia, obstruction of the ductal system is responsible for approximately 40% of cases [1]. Men with obstructive azoospermia may produce a pregnancy either by: surgical correction of the obstruction, which may produce pregnancy by intercourse and obviate the need for assisted reproductive technology; retrieval of sperm from the male s reproductive system for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). Testicular sperm retrieval and its use in the treatment of infertile couples was performed for the first time in 1995 [2]. Sperm can be obtained either aspirated by puncture from the epididymis (PESA percutaneous epididymal sperm aspiration) or testicle (TESA testicular sperm aspiration), or extracted from the testicle by biopsy [3]. Micro-dissection sperm retrieval (TESE testicular sperm extraction) performed with an operative microscope is generally considered to be the best method with higher spermatozoa retrieval rates and minimal tissue loss in azoospermic patients [4]. At present, there are no options for men with azoospermia and failed TESE to have biological children [5]. Since sperm cells retrieval by these invasive methods comes from subjects with important sperm pathology, the obtained sperm cells are extremely few and show major changes in motility and morphology. Oocyte penetration is possible with intracytoplasmic sperm injection (ICSI) techniques. The head of the sperm that contains the cell nucleus and the entire genetic information is injected inside the oocyte using micropipettes handled by a special micromanipulation system attached to an inverted microscope [6]. However, the low magnification used in routine ICSI microscopy ( 200, 400) results in limitations in identifying sperm organelle malformations, particularly vacuoles in the sperm head, where major defects, such as abnormal sperm head size proportions and midpiece abnormalities were observed [7]. For a better morphology assessment of mobile spermatozoa, in the last years there has been imagined a new technique for selecting sperm. Bartoov et al. have developed a new method known as motile sperm organelle morphology examination (MSOME), which evaluates the real-time movement of sperm [8, 9]. They made it possible to obtain a sperm with a normal nucleus by combining MSOME and micromanipulation technology. This process is known as the modified IVF process or intracytoplasmic ISSN (print) ISSN (online)

2 880 Constantin-Cristian Văduva et al. morphologically selected sperm injection (IMSI) because only one moving sperm is selected when performing ICSI using MSOME [7]. IMSI uses a high microscope magnification 6000 and resolution is associated with better embryo quality and higher rates of implantation and pregnancy [10]. IMSI procedure is a good option for couples with a first unsuccessful ICSI cycle [11]. In this article, we present a successful case of TESE followed by IMSI and ICSI in a couple of male infertility due of obstructive azoospermia. The procedure was performed in 2015 and was completed with the birth of a healthy baby in time. degenerate, spermatogonia cells, primary spermatocytes, very rare elongated spermatids and sperms. Case presentation The couple attended for fertility examination reporting a history of 10 years of infertility. Age of the patient was 31 years old. He was diagnosed with azoospermia by performing spermiograms in four different laboratories. No other pathology was present in the male. Urological examination was normal. Obstructive azoospermia was diagnosed by evaluating hormonal status of the patient that revealed normal values: FSH (follicle-stimulating hormone) 4.26 IU/L, LH (luteinizing hormone) 6.32 IU/L, testosterone ng/ml, TSH (thyroid-stimulating hormone) 2.5 IU/L and prolactin 668 miu/l. Sperm and urethral cultures were normal. The sperm antibodies were absent to both partners. The genetic examination showed normal karyotype with absence of micro-deletions on the Y chromosome or mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. The woman was 29 years old whose hormonal status indicated an increased ovarian reserve on day 2 of the cycle: FSH 6.5 IU/L, LH 13.3 IU/L, E2 (estradiol) 53 ng/ml, AMH (anti-müllerian hormone) 4.6 ng/ml. There were no clinical features of polycystic ovarian syndrome. An informed consent was obtained for treatment procedure. The woman received controlled ovarian stimulation that lasted 11 days. The ovarian stimulation was done with the antagonist protocol and using 20 ampoules of 75 IU of FSH + LH (Menopur, Ferring) and six ampoules of Ganirelix (Orgalutran, MSD). Ovulation was triggered with 250 μg rhcg (recombinant human chorionic gonadotropin Ovitrelle, Serono). They were obtained nine oocytes, which had undergone in vitro fertilization procedure. The urologist performed testicular biopsy by classic technique under intravenous anesthesia, making unilateral hemiscrotomy with minimal incision and testicular tissue sampling. The test was carried out by micro-dissection of the testis. A piece of testicular tissue was investigated for histopathological changes (Figure 1). Harvested tissue was fixed in 10% buffered formalin solution and embedded in paraffin. Serial sections were made with a thickness of 3 μm. These were stained with Hematoxylin Eosin (HE) and examined at the optical microscope at 100 magnification. The pathologist observed discreet interstitial fibrosis with mild thickening of the seminiferous tubules own tunic. It also observed an appreciable reduction in the number of germ cells in some areas disorganized and Figure 1 Testicular tissue obtained by TESE. Rare seminiferous tubules, which present advanced sclerosis correlated with appreciable reduction of germ cells (HE staining, 100). Another piece of testicular tissue was analyzed by the embryologist. Perioperative microscopically examination of a wet preparation of the shredded specimens was performed at 400 ICSI magnification according to a technique described by Jow et al. [12]. The embryologist noted the existence of a very small number of morphologically normal spermatozoa that had minimal motility. There was observed an elevated proportion of immature germ cells belonging to immature germs lines: spermatogonia, spermatocyte and spermatid. Mature forms showed abnormal morphology of the head and neck of sperm. The embryologist excluded from ICSI spermatozoa exhibiting severe head defects, which were clearly seen at this magnification: pin, amorphous, tapered, round, and multinucleated head (Figure 2). Figure 2 Testicular tissue obtained by TESE. We notice the presence of sperm cells from germ-line and rare mature forms represented by sperm with atypical forms (freshly prepared, 400). Few mature spermatozoa were selected by IMSI under high magnification using an inverted microscope Nikon Eclipse Ti-U equipped with modular contrast Hoffman and micromanipulation system Narishige. This system is adapted for the observation and manipulation of living cells; it is equipped with a transparent thermoplate at 370C (Tokai Hit). The microscope incorporated a 60 air objectives with modulation contrast illumination (RI IMSI,

3 Pregnancy resulting from IMSI after testicular biopsy in a patient with obstructive azoospermia RI, Cornwall, UK). The images were captured using a high-resolution DC2 IMSI camera (Nikon) with 2/3 CCD and an ultra-high resolution 28 color video monitor. Calculation of the total magnification on the monitor were based on three parameters: (1) objective magnification 60; (2) magnification selector 1.5 and (3) (a) CCD chip diagonal dimension 11 mm and (b) video monitor diagonal dimension mm, for a calculated video magnification (b/a) of Thus, total magnification = microscope magnification ( 90) video magnification ( 64.65) = There were selected nine sperm cells presenting morphological IMSI criteria of normality. Spermatozoa were considered morphologically suitable for ICSI procedures when the head of the spermatozoa was a regular oval shape, μm in length and μm in width, and was characterized by a maximum of a single vacuole not more than 4% the total area of the sperm head; the midpiece was also required to be regular, rectangular shape of between 4.0 and 5.0 μm in length (Figure 3). Figure 3 Sperm selected by IMSI with relative normal morphology (freshly prepared, 900). The selected spermatozoa were used for the ICSI procedure and all nine were injected in oocytes. Nineteen hours after injection, six oocytes had good fertilization pattern with two pronucleus and two polar bodies well defined. These were incubated for 72 hours, at 370C, in reduced oxygen culture condition: 6% CO2, 5% O2, and 89% N2, in a bench-top incubator (MINC, Cook) using specific cell culture media (MediCult, Denmark). There were obtained six normal embryos: five 8 10 cell embryos without fragmentation, well-defined blastomeres and few compactions and one five-cell embryo with few fragmentations. Three embryos were transferred in utero and three embryos were frozen (Figure 4). Figure 4 Three viable embryos of 8 10 cells with normal morphology and no fragmentation 72 hours after fertilization by ICSI injection of oocyte with selected sperm. These embryos were transferred in utero (freshly prepared, 200). 881 A singleton pregnancy was achieved. At the end of 2015, after an uneventful pregnancy, patient delivered at 39 weeks. The newborn had 3100 g in weight, 53 cm in length and no medical problem. Discussion Infertility is defined as the failure of a couple to become pregnant after one year of regular, unprotected, sexual intercourse. However, most infertile couples, and particularly men, are reluctant to use donor sperm [3]. Currently, despite severe male factor infertility, pregnancy may still be achieved by current developed technology. Among the causes of male infertility, treatment of azoospermia is a challenge for any specialist in infertility. To determine that a semen sample is truly azoospermic, centrifugation of the semen sample with meticulous microscopic examination of the pellet is necessary. For all patients with azoospermia, a complete history and physical examination is necessary to identify potentially correctable causes of male factor infertility [1]. Today exciting advances in male infertility have introduced innovative therapeutic options. Microsurgical testicular sperm retrieval techniques, offers to infertile couples, including those with no sperm azoospermia, a greatly improved chance to conceive their own biological offspring [13]. Testicular biopsy seems to be one of the most feasible techniques obtaining spermatozoa from azoospermic men because a single biopsy of testis may provide either spermatozoa if the specimen contains sperm or spermatids in case when harvested sperm do not contain any sperm at the moment of insemination [6]. The introduction of intracytoplasmic sperm injection procedures into the techniques of assisted reproduction marked an important milestone in the development of the field [14]. With this procedure, patients whose semen samples were insufficient for intrauterine insemination or in vitro fertilization techniques could achieve pregnancy using micromanipulation techniques [15]. Spermatozoa morphology is the best selection criteria for intracytoplasmic injection. Several studies demonstrated a positive correlation between optimal sperm morphology evaluation and ICSI outcomes improvement. De Vos et al. evaluated the impact of individual sperm morphology on ICSI outcome: fertilization, embryo development and implantation rate [16]. This study was performed by using an inverted light microscope and sperm cells were classified as normal in accordance to Kruger et al. criteria [17]. The authors demonstrated that the injection of abnormal spermatozoa was associated to a significantly lower fertilization, implantation and pregnancy rate when compared to ICSI cycles performed by injecting spermatozoa with apparently normal morphology. The low magnification and resolution of the microscope used for the morphology assessment represented the main limitation of this study. However, due to relatively low power magnification ( 200/ 400), ICSI technique has its limits in terms of changes in intracellular characterization [18]. IMSI technique currently is the only real-time microscopy procedure who does not require fixation and who can select viable sperm with minimal ultrastructural defects [7]. Indeed, most of the enzymatic or genetic tests currently available cannot be performed on viable, unfixed,

4 882 spermatozoa. The detection of large nuclear vacuoles at high magnification could be related to DNA fragmentation and denaturation. It is widely accepted that DNA integrity plays an important role in the fertilization process and in the development and implantation of embryos [19, 20]. Furthermore, Vanderzwalmen et al. reported a negative influence of spermatozoa with large nuclear vacuoles in the head on the capability of embryos to develop to the blastocyst stage [21]. Antinori et al. pointed out the need to increase the efficiency of micro-insemination techniques especially in those countries, such as Italy, where the law limits the number of fertilizable oocytes [22]. Several studies demonstrated that IMSI provided positive results in couples with severe male factor infertility or repeated ICSI failures [8, 23, 24]. In all these studies, the clinical pregnancy rate was noticeably improved in the IMSI group. Moreover, the IMSI group result was associated with a lower abortion rate [10, 17]. IMSI technique for selecting sperm morphology has proved useful in choosing cells with maximum potential to give rise to viable and good quality embryos, considering that their harvest was performed by microdissection of testicular biopsy tissue [9]. The number, mobility, maturity and sperm morphology was negatively influenced by intrinsic testicular pathology and invasive microsurgery technique followed by microdissection [8]. Obviously, every single good-quality oocyte deserves the best spermatozoon available in the sperm sample to be use for microinjection, in order to obtain the highest probability of developing a high quality embryo that implants. It needs to be defined what is absolutely needed in terms of nuclear normalcy presenting with no single vacuole in the sperm head on the one hand and what is at least as good or good enough to be used as second-best spermatozoon on the other hand without compromising oocyte fertilization, embryo development and implantation potential. Using testis biopsy sperm for azoospermia may gain the same high pregnancy rate as the normal ejaculation sperm, but efficiency of spermatid application is doubted because low fertilization and pregnancy rates influence seriously its success. As cell-cloning technology develops, other options for treatment of azoospermic men include use of secondary spermatocytes, sperm cell cloning, and artificial sperm production by inducing stem cells and adult somatic cells into sperm cells. Although these technologies still remain at animal research stage and demonstrate a low efficiency, we believe that application of these novel techniques will greatly improve chance for infertile couples (particular azoospermic men) to conceive their own biological offspring in the next decade. IMSI is a promising new technique that may help to improve laboratory and clinical performance in assisted reproduction. In cases of azoospermia, the combination of ICSI with getting sperm through TESE represents an edge therapy of male infertility. In our case, the embryos quality obtained was demonstrated by the birth of a healthy term baby. After our knowledge, this is the first case in Romania of pregnancy obtained through in vitro fertilization technique IMSI with ICSI on the sperm achieved by testicular biopsy. Constantin-Cristian Văduva et al. Conclusions The ICSI method involves a magnification of enabling a relatively coarse selection. The embryologist excludes from ICSI spermatozoa exhibiting severe morphological defects, which are clearly seen at this magnification. The selection with IMSI is performed under ultra-magnification ( 6000) with possibility to detect the ultrastructure of the head, midpiece and its organelles. This new technique has a theoretical potential to improve reproductive outcomes among couples undergoing assisted reproduction techniques. In our case, IMSI help us to select the best spermatozoa in condition in which the number, mobility, maturity and morphology of these germinal cells were negatively influenced by intrinsic testicular pathology and invasive microsurgery technique followed by micro-dissection. In light of improved pregnancy rate and delivery rate, we tend to recommend promoting the IMSI method for couples with obstructive azoospermic male infertility. Further trials are necessary to improve the evidence quality before recommending IMSI in clinical practice. Conflict of interests The authors declare that they have no conflict of interests. Consent Written informed consent was obtained from the couple of patients for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of Rom J Morphol Embryol. References [1] Jarow JP, Espeland MA, Lipshultz LI. Evaluation of the azoospermic patient. J Urol, 1989, 142(1): [2] Devroey P, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, Van Steirteghem A, Silber S. Pregnancies after testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) in non-obstructive azoospermia. Hum Reprod, 1995, 10(6): [3] Craft I, Tsirigotis M. Simplified recovery, preparation and cryopreservation of testicular spermatozoa. Hum Reprod, 1995, 10(7): [4] Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod, 1999, 14(1): [5] Gassei K, Orwig KE. Experimental methods to preserve male fertility and treat male factor infertility. Fertil Steril, 2016, 105(2): [6] Van Steirteghem AC, Liu J, Joris H, Nagy Z, Janssenswillen C, Tournaye H, Derde MP, Van Assche E, Devroey P. Higher success rate by intracytoplasmic sperm injection than subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum Reprod, 1993, 8(7): [7] Kim HJ, Yoon HJ, Jang JM, Oh HS, Lee YJ, Lee WD, Yoon SH, Lim JH. Comparison between intracytoplasmic sperm injection and intracytoplasmic morphologically selected sperm injection in oligo-astheno-teratozoospermia patients. Clin Exp Reprod Med, 2014, 41(1):9 14. [8] Bartoov B, Berkovitz A, Eltes F, Kogosovsky A, Yagoda A, Lederman H, Artzi S, Gross M, Barak Y. Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertil Steril, 2003, 80(6): [9] Bartoov B, Berkovitz A, Eltes F, Kogosowski A, Menezo Y, Barak Y. Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome. J Androl, 2002, 23(1):1 8.

5 Pregnancy resulting from IMSI after testicular biopsy in a patient with obstructive azoospermia 883 [10] Hazout A, Dumont-Hassan M, Junca AM, Cohen Bacrie P, Tesarik J. High-magnification ICSI overcomes paternal effect resistant to conventional ICSI. Reprod Biomed Online, 2006, 12(1): [11] Klement AH, Koren-Morag N, Itsykson P, Berkovitz A. Intracytoplasmic morphologically selected sperm injection versus intracytoplasmic sperm injection: a step toward a clinical algorithm. Fertil Steril, 2013, 99(5): [12] Jow WW, Steckel J, Schlegel PN, Magid MS, Goldstein M. Motile sperm in human testis biopsy specimens. J Androl, 1993, 14(3): [13] Tesarik J, Mendoza C. Treatment of severe male infertility by micromanipulation-assisted fertilization: an update. Front Biosci, 2007, 12: [14] Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 1992, 340(8810): [15] Palermo GD, Cohen J, Alikani M, Adler A, Rosenwaks Z. Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil Steril, 1995, 63(6): [16] De Vos A, Van De Velde H, Joris H, Verheyen G, Devroey P, Van Steirteghem A. Influence of individual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection. Fertil Steril, 2003, 79(1): [17] Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA, Smith K. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril, 1986, 46(6): [18] Marci R, Murisier F, Lo Monte G, Soave I, Chanson A, Urner F, Germond M. Clinical outcome after IMSI procedure in an unselected infertile population: a pilot study. Reprod Health, 2013, 10:16. [19] Franco JG Jr, Baruffi RL, Mauri AL, Petersen CG, Oliveira JB, Vagnini L. Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI. Reprod Biomed Online, 2008, 17(1): [20] Garolla A, Fortini D, Menegazzo M, De Toni L, Nicoletti V, Moretti A, Selice R, Engl B, Foresta C. High-power microscopy for selecting spermatozoa for ICSI by physiological status. Reprod Biomed Online, 2008, 17(5): [21] Vanderzwalmen P, Hiemer A, Rubner P, Bach M, Neyer A, Stecher A, Uher P, Zintz M, Lejeune B, Vanderzwalmen S, Cassuto G, Zech NH. Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles. Reprod Biomed Online, 2008, 17(5): [22] Antinori M, Licata E, Dani G, Cerusico F, Versaci C, d Angelo D, Antinori S. Intracytoplasmic morphologically selected sperm injection: a prospective randomized trial. Reprod Biomed Online, 2008, 16(6): [23] Berkovitz A, Eltes F, Lederman H, Peer S, Ellenbogen A, Feldberg B, Bartoov B. How to improve IVF-ICSI outcome by sperm selection. Reprod Biomed Online, 2006, 12(5): [24] Berkovitz A, Eltes F, Yaari S, Katz N, Barr I, Fishman A, Bartoov B. The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Hum Reprod, 2005, 20(1): Corresponding author Constantin-Cristian Văduva, Senior Lecturer, MD, PhD, Department of Obstetrics and Gynecology, University of Medicine and Pharmacy of Craiova, Emergency County Hospital of Craiova, 1 Tabaci Street, Craiova, Romania; Phone , hitmed@gmail.com Received: February 26, 2016 Accepted: September 7, 2016