First Romanian live birth after Preimplantation Genetic Testing in a couple with Severe Oligospermia determined by Y Chromosome Microdeletions

Y chromosome microdeletions (YCM) represent a genetic disorder, frequently responsible for the disruption of the spermatogenesis process. Its prevalence is about 20% among patients diagnosed with oligozoospermia. Here we report the successful live birth of a female baby after morphologically selected sperm injection (IMSI) and PGD for a couple with severe oligospermia related to Y chromosome microdeletions. Blood samples from the couple were obtained and the cytogenetic analysis was carried out on in vitro cultured peripheral lymphocytes. DNA extraction and molecular analysis performed on blood samples from the male patient using multiplex-PCR method enabled the identification of 24 microdeletions in AZFa, AZFb or AZFc loci, the absence of a series of markers being observed. The couple underwent an IVF cycle associated with PGD. The resulting embryos were cultured until day 3 and subsequently, the blastomere biopsy was carried out on 7 of the viable embryos. The entire genome was scanned using the Microarray-CGH technique with the Agilent GenetiSure Pre-Screen test. The analysis of the 7 embryos obtained through IMSI revealed an affected genotype in four of biopsies and a non-affected genotype in three biopsies. Two of the unaffected embryos were female and one was male. Accordingly, it was decided to carry out the transfer of the most suitable female embryo, which resulted in the birth of a healthy baby. The results confirm the importance of performing these type of genetic investigations in patients with a history of male infertility with genetic component.


Introduction
Preimplantation genetic diagnosis (PGD) is a technique performed in conjunction with in vitro fertilisation (IVF), dedicated at identifying possible genetic defects in embryos obtained through human assisted reproductive technology (ART) [1]. Usually, PGD is performed for couples in which one or both partners suffer from a genetic disorder. Therefore, embryos may be tested to reveal whether they are affected by the same abnormalities. Over 350 different medical conditions may be diagnosed, with an accuracy of about 99.5% [2].

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Unlike other well-known methods of prenatal diagnosis (e.g. amniocentesis), which is only available when the pregnancy is established, PGD is performed before implantation. It can aid both medical team and the patients in choosing the most genetically suitable embryo(s). In contrast to amniocentesis, where the parents may risk being faced with the dramatic decision to terminate an ongoing pregnancy, PGD may improve the likelihood of obtaining a normal pregnancy [3].
The successful implementation of PGD technique in an IVF clinic involves collaboration between several areas such as IVF, genetics and single cell diagnosis laboratories.
Notwithstanding, the ability of the PGD test to enable parents affected by a genetic disease to have a healthy child, there are some legal, ethical and medical implications in Romania which were highlighted before [4], but unfortunately still remain unanswered.

Material and Methods
A 28 years old female patient and her 30 years old male partner presented for fertility advice after unsuccessfully trying to conceive for almost 2 years. The initial consultation aimed at assessing the medical history of the patients, the eventual risk factors, as well as the results of previous investigations. Both patients were submitted to a viral screening for hepatitis and HIV (Ag HBs, Ac HBc, Ac HCV, Anti HIV1+ 2, VDRL), both patients being tested negative.    The oocyte retrieval was carried out 36 hours afterwards.
Subsequently, the oocytes were inseminated using IMSI and the resulting embryos were cultured and monitored until day 3 using a time-lapse system (EmbryoScope, Vitrolife, Denmark). On day 3, the blastomere biopsy was carried out by an embryologist and 7 embryos were biopsied.

Results and Discussion
The analysis of the 7 embryos revealed an affected genotype in 4 of biopsies and a non-affected genotype in 3 of the biopsies. Two of the unaffected embryos were female and one was male ( Figure   1,4,6).

Am J Biomed Sci & Res
Copy@ Ovidiu-Dumitru Ilie It has been shown that Y chromosome related infertility is characterised by azoospermia (absence of spermatozoa) or different forms of oligozoospermia (reduced number of spermatozoa) [5]. According to a study conducted in Romania, the incidence of genetic defects is higher in infertile patients diagnosed with severe oligospermia [6].
Over 60% of microdeletions observed in the structure of the Y chromosome are located in the AZFc region. Since this region is not close to the center of the Y chromosome as the AZFa or AZFb regions, the microdeletions at this level have a less profound impact on sperm production and thereby, some patients diagnosed with AZFc deletions present with a reduced total number of spermatozoa (oligospermia) [7].
The deletions in the AZFc region of the Y chromosome are the most frequent cause of spermatogenic failure that can be defined molecularly [8]. In a couple of studies, it has been revealed that the AZFc deletions are responsible for almost 20% of cases with nonobstructive azoospermia or severe oligozoospermia [9,10].
Moreover, a recent study revealed that the development of embryos obtained through ICSI (Intracytoplasmic Sperm Injection) from partners diagnosed with AZFc microdeletions and from those without microdeletions was comparable, suggesting that AZFc microdeletions would not affect the outcome of ICSI procedure [11].
Since female offspring inherit only the X chromosome from the father, they are not at risk. It has been suggested that in IVF therapy for couples where the male has a Y deletion, PGD is recommended in order to establish the gender of the embryos and to clarify the presence of the Y chromosome microdeletions [5].
Surprisingly, two recent studies highlighted the probability of obtaining female embryos is higher when using IMSI (Intracytoplasmic Morphologically Selected Sperm Injection) compared to ICSI (Intracytoplasmic Sperm Injection). Therefore IMSI was considered to be the method of choice in the case of this couple [12,13].
Preimplantation screening aCGH technique is a valuable technique. Although, there are certain considerations that must be taken into consideration. Since the biopsy is performed using a single cell (third day biopsy) or from a small group of trophoectoderm cells (fifth day biopsy), test results may offer an altered image of the entire embryo.

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However, the identification of major chromosomal abnormalities such as trisomy or monosomes (aneuploidy) in embryos with good or very good morphology and with satisfactory evolution confirms that preimplantation genetic testing has a defining role in the right and informed choice of the appropriate embryo.
We also have to keep in mind that interpreting the results of aCGH tests should not be rigid, small anomalies that cannot be linked in a clinical context may actually be technical artifacts as

Conclusion
In the era of human assisted reproduction, the genetic screening has opened new horizons. Thereby the preimplantation testing should be integrated with other IVF techniques as a companion test that may complement the overall picture and help in making the best decisions in the task of obtaining healthy offspring.
The Y chromosomal deletion assays provides a precise diagnostic, thus offering clinicians the chance to establish an appropriate treatment plan and last but not least to better estimate the couple's chances of conceiving.
In this case, the PGD procedure enabled the identification of 4 embryos with affected genotype and additionally the sex of all the embryos was established. Therefore the medical team decided to transfer a female embryo to the patient since the microdeletions in the AZFc region of the Y chromosome is a genetically transmitted disorder and is one of the most frequent causes of spermatogenic failure.
To our knowledge, this is the first reported case of IMSI However, almost 20 years after the creation of the Eshre PGD consortium, preimplantation genetic testing is still inaccessible to many couples because of the extremely high costs that in many countries, including Romania, are not reimbursed through the public health system. Future challenges are related to the development of new molecular diagnostic methods, but also the necessity of a wellestablish strategy that would ensure accessibility to combined IVF and preimplantation genetic testing for patients through national health programs.