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Revista Română de Anatomie funcţională şi clinică, macro- şi microscopică şi de Antropologie

Vol. XIV – Nr. 2 – 2015 CLINICAL ANATOMY

BIRTH OF HEALTHY TWINS AFTER TESTICULAR BIOPSY WITH INTRACYTOPLASMATIC SPERM INJECTION FROM

A PATIENT WITH NON-OBSTRUCTIVE AZOOSPERMIA

Raluca Tulin1,2, Alice Albu2,3, I. Bulescu1, O. Munteanu1, T. Leonard2, D. Albu2,4

University of Medicine and Pharmacy “Carol Davila”, Bucharest1. Faculty of MedicineDepartment of Anatomy3. Faculty of Medicine

Department of Endocrinology4. Faculty of Dental Medicine

Department of Obstetrics and Gynecology2. MedLife Memorial Hospital, Bucharest

Department of Materno Fetal and Human Reproduction

BIRTH OF HEALTHY TWINS AFTER TESTICULAR BIOPSY WITH INTRACYTOPLAS-MATIC SPERM INJECTION FROM A PATIENT WITH NON-OBSTRUCTIVE AZOOSPERMIA (Abstract): Non-obstructive azoospermia used to be considered an untreatable cause of male in-fertility. By the microinjection technique, sperm that has been surgically extracted from the testis can be injected into the oocyte cytoplasm to obtain an embryo. Severe asthenozoospermia, severe oligozoospermia and azoospermia have all been success fully treated by using the ICSI tech-nique. Sperm retrieval in combination with ICSI is the only medical procedure for an azoospermic man to father a child. Different techniques, especially testicular sperm extraction (TESE), have evolved over time and have dramatically improved the outlook for men with testicular azoosperm-ia. However sperm retrieval rates are associated not only with the operation proposed but espe-cially with a distinct pattern of prognostic factors that must be effectively managed for all these infertile patients for their best benefit. This report is on a case of non-obstructive azoospermia that was successfully treated by testicular biopsy (TESE) with ICSI procedure in private Hospital, MedLife Bucharest. The result is the birth of 2 baby-boys perfectly healthy. Key words: INFER-TILITY, ICSI (INTRACYTOPLASMATIC SPERM INJECTION), AZOOSPERMIA

INTRODUCTIONThirty seven years ago, the world’s first

“baby tube” conceived by the in vitro fertiliza-tion (IVF) technique, Louise Brown, was born. Since then, a huge progress has been made in the IVF techniques. Of all advancements, the most significant are the micromanipulation techniques that can solve female but especially male fertil-ity problems. Intracytoplasmic sperm injection (ICSI) was first described in 1992 by Palermo and all. and has changed the management of severe male infertility (1). Since 1992, severe asthenospermia, severe oligospermia and azoo-spermia (obstructive or non-obstructive) have all been success fully treated by using the ICSI technique (2,3).

Later, in 1995 Vanderzwalmen reported a case of succesfull fertilsation of a human oocyte by a late stage spermatid with intracytoplas-matic sperm injection (4,5). Today it is well established that mature testicular spermatozoa could be found in men with non-obstructive azoospermia (6,7).

Thus, testicular sperm extraction (TESE) combined with intracytoplasmic sperm injec-tion (ICSI) offer azoospermic patients the pos-sibility of fathering their own genetic children even if they do not have normal spermatogen-esis. TESE and ICSI have become the standard protocols for treatment of patients with func-tional azoospermia who have no source of sper-matozoa except the testis (2).

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We report a case of non-obstructive azoo-sper mia that was successfully treated by a com-bination of ICSI with TESE (Testicular Sperm Extraction), the result being birth of two healthy boys.

CASE REPORTA 33-year-old man and 32-year-old woman

who had no significant family or medical prob-lems presented to the IVF Centre MedLife Me-morial Hospital in January 2014. The man was found to be azoospermic from three different semen analyses. The physical examination showed normal secondary sexual characteristics; the testes, epididymides, and vasa were normal. The level of follicle-stimulating hormone was 14.64 mIU/mL (normal range 0.95-11.95 mIU/mL). The ultrasound of the testes showed a slightly bilateral hypotrophy (Fig. 1.A and B). The couple was counseled about and subsequent-ly consented to the ICSI procedure and the need to recover sperm by performing a testicular biopsy (TESE). The investigations of the woman showed a normal uterine cavity and uterine tubes (hys-terosalpingography was performed) with a de-creased ovarian reserve (AMH = 0.9 ng/ml with normal range 1-8 ng/ml). The woman’s hor-mone profile on the third day of the cycle was found to be normal. The chromosome analysis of the peripheral leukocytes was normal for both patients. After counseling, the couple de-cided to have ICSI with testicular biopsy.

In April 2014, controlled ovarian hyperstim-ulation was achieved by giving the woman in-tramuscular human menopausal gonadotropin, HMG 150 U twice daily and human follicle stimulating hormone (preparation of recombi-nant DNA origin) r-hFSH 150 UI daily, for 10 days. Previously was performed a pituitary sup-pression using triptorelinum that was started on

day 1 of the menstrual cycle. Follicular growth was monitored by determining the plasma oestradiol level and by intravaginal ultrasonog-raphy. At the end of the stiumulation protocol the luteinisation was induced by human chori-onic gonadotrophin (hCG) 10000 U that was given by intramuscular injection and oocyte collection was performed 36 hours later under guidance of vaginal ultrasound.

In the same day with the ovarian retrieval an open testicular biopsy under general anaesthe-sia was performed on the man by a urologist surgeon. The biopsy tissue was transferred to a petri dish and shredded into small pieces us-ing. The suspension was then examined under the microscope and when sperm were identi-fied, the biopsy tissue was removed and the suspension was centrifuged. After the sperm pellet was re suspended, approximately 50 live, normal-looking mature sperm were identified.

Oocyte collection was performed by a gy-necologist surgeon while the woman was se-dated. By using an ultrasound-guided trans-vaginal needle aspiration technique, 4 oocytes were obtained. They were examined and were all found to be in the metaphase II stage of meiosis and suitable for ICSI. Fertilisation was checked at 16 hours after injection and found all of them fertilized.Fertilisation was assessed as normal when two clearly distinct pronuclei were present. The state of embryo cleavage and quality was assessed after further 24 hours of in vitro cultured. The embryos were evaluated according blastomere size equality and the relative proportion of anucleate fragments. On day 5 were transferred inside female uterine cavity 2 blastocysts (Fig. 2 A and B).

Pregnancy was confirmed 14 days after the procedure and progressed normally. At 14 weeks of gestation, an ultrasound was performed and

Fig. 1. Testicular ultrasound with slightly bilateral hypotrophy (A and B)

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showed the two babies boys to be normal. Preg-nancy progressed without complication but at 33 weeks the two boys were delivered by a caesarian section.

DISCUSSIONIf in the past azoospermia was synonymus

with sterility and the only available options were to adopt children or to use sperm cells from a donor, today we have a lot of tools to perform artificial insemination of the oocyte using the new methods of ICSI with sperm cells obtained directly by a testicular biopsy.

Experience around the world has shown that in most men who present with non-obstruc-tive azoo- spermia for testicular sperm extrac-tion, the testes have islets of normal spermato-genesis in the semi- niferous tubules and thus contain normal sperm cells (8).

Unfortunately, the number of normal sperm is usually insufficient for IVF, which requires 50000 motile sperm. Allied procedures are thus needed. Intra- cytoplasmic sperm injection has brought hope to a situation where “the wife is producing more eggs than the husband’s sper-matozoa” (9). This situation occurred on this couple. Intracytoplasmatic sperm injection ia an effective remedy for many types of male factor infertility (1).

Our days many other procedures of micro-manipulation and transfer were developed and we have a lot of new tools for treatment of couple infertility. Micromanipulation followed by immediate fallo- pian tube transfer (MIFT) was first reported in 1994 by McLachlan et al who replaced oocytes after performing sub-zonal sperm injection (10).

Treating infertility by using assisted repro-duction techniques such as gamete (sperm and

egg) intrafallopian transfer (GIFT) may be bet-ter than using IVF, because of the more physi-ological tubal environment for fertilisation and embryo develop- ment (10).

Sperm retrieval can be achieved by open testicular biopsy (TESE), microtesticular sperm extraction (micro-TESE) or by epididymal as-piration (2,8). Among men with non-obstruc-tive azoospermia who agree to undergo testicu-lar biopsy, some will be found to have no sperm. At present there is no way to predict which man will have no sperm. When sperm are found, clinical pregnancies can occur for half (5/10) of these couples using TESE with ICSI, with ongoing pregnancy and delivery for 4 of 10 (40%). Many men with non obstructive azoo-spermia will have retrievable sperm with testis biopsy that are suitable for ICSI; however, 6 of 16 (38%) couples will not have sperm re-trieved with TESE and may undergo an unnec-essary IVF procedure (11). Couples should be prepaired for the possibility that no sperm will be recovered and consider the use of donate sperm if necessary.

CONCLUSIONWe report a case with twins pregnancie in a

couple where the husband has non-obstruc-tive azoospermia. Using testicular sperm ex-traction and a direct ICSI technique the woman became pregnant. The result was delivery of a two healthy, normal baby boys in November 2014. Testicular sperm retrieval and assisted reproduction techniques may enable such cou-ples to conceive. We consider that as in all cases of severe male-factor infertility requiring intracytoplasmatic sperm injection, genetic screening and prenatal testing should be strong-ly recommended.

Fig. 2. The 2 blastocysts that were transferred (A and B)

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