The evolving role of genetic tests in reproductive medicine

REVIEW

The evolving role of genetic tests

in reproductive medicine

Federica Cariati

_{, Valeria D’Argenio}

_{and Rossella Tomaiuolo}

Abstract

Infertility is considered a major public health issue, and approximately 1 out of 6 people worldwide suffer from infertility during their reproductive lifespans. Thanks to technological advances, genetic tests are becoming increas-ingly relevant in reproductive medicine. More genetic tests are required to identify the cause of male and/or female infertility, identify carriers of inherited diseases and plan antenatal testing. Furthermore, genetic tests provide direc-tion toward the most appropriate assisted reproductive techniques. Nevertheless, the use of molecular analysis in this field is still fragmented and cumbersome. The aim of this review is to highlight the conditions in which a genetic evaluation (counselling and testing) plays a role in improving the reproductive outcomes of infertile couples. We conducted a review of the literature, and starting from the observation of specific signs and symptoms, we describe the available molecular tests. To conceive a child, both partners’ reproductive systems need to function in a precisely choreographed manner. Hence to treat infertility, it is key to assess both partners. Our results highlight the increasing importance of molecular testing in reproductive medicine.

Keywords: Genetic tests, Reproductive medicine, Male infertility, Female infertility, Assisted reproductive technology

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,

and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/

publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Introduction

During the last few decades, there have been a series of striking advancements in reproductive and laboratory medicine that have essentially caused these two fields to become inextricably connected. Laboratory medicine now plays a critical role in all stages of the reproductive process, from diagnostic approaches to the choice of the most complex therapy.

In particular, genetic tests are carried out for three main purposes in reproductive medicine: the identifica-tion of the infertility causes, identificaidentifica-tion of genetic dis-eases transmissible to offspring, and optimization of the assisted reproductive technology (ART) (Fig. 1).

The overall fertility rate is decreasing; for example, in the US, 12% of women receive fertility treatment over the course of their lifetimes, so it is important to empha-size the fertility journey of couples [1]. The reproduc-tive systems of both partners function in a combined

and precisely coordinated way to conceive a child; for this reason, evaluation of both members of the couple is mandatory.

A medical evaluation is indicated when the couple fails to achieve pregnancy after 12  months of regular, unprotected sexual intercourse [2]. Currently, the diag-nostic timeline of infertile couples includes biochemical and instrumental analyses that allow for a diagnosis in 65% of cases; in the remaining 35% of cases, which are undiagnosed, genetic tests are performed. Considering that approximately 15% of genetic disorders are asso-ciated with infertility and that similar clinical signs can have genetic and nongenetic causes, it is important that an infertility diagnosis be determined by the combina-tion of an accurate medical history and instrument- and laboratory-based evaluations, including targeted genetic tests [3]. Confirmation of the clinical diagnosis through genetic evaluation (counselling and testing) can lead to more specific and targeted medical management.

In addition, genetic tests are also indicated for the identification of genetic diseases that are transmis-sible to the offspring: preconception screening allows couples who are planning to become pregnant to know

Open Access

*Correspondence: dargenio@ceinge.unina.it

2 _{Dipartimento di Medicina Molecolare e Biotecnologie Mediche,}

Università degli Studi di Napoli Federico II, Via Sergio Pansini 5, 80131 Naples, Italy

their reproductive risk a priori. Normally, gametes with genetic or chromosomal alterations have reduced repro-ductive potential. Thanks to ART, many of these difficul-ties can be overcome, and therefore, genetic tests (carrier screening, preimplantation and prenatal diagnosis) have

the crucial impact of monitoring the possible transmis-sion of these genetic alterations to the offspring [4, 5].

Another application field of molecular diagnostics is related to the antenatal diagnosis. To date, the diagnos-tic options for couples at risk of transmitting a specific Fig. 1 The three main fields of application for which genetic testing is required to improve reproductive medicine: identification of the infertility causes (a), identification of genetic diseases transmissible to offspring (b), and optimization of the assisted reproductive techniques (c)

inherited disorder to their offspring are preimplanta-tion genetic testing (PGT) and prenatal diagnosis (PND). These two diagnostic procedures share the same pur-pose but differ in diagnostic time, type of sampling, and laboratory procedures. In addition to the more tradi-tional laboratory investigations, it is now undisputed that molecular biology methods for PGT support the efficacy of ART techniques, contributing significantly to their success (reductions in time, effort and cost) [5, 6].

To optimize the application of genetic tests in clinical practice, in this review, we discuss (1) the genetic condi-tions related to infertility, including the common and rare ones that are case appropriate; (2) the diagnostic strate-gies in families at risk of known monogenic disease trans-mission; and (3) the impact of PGT in the optimization of ART techniques.

Materials and methods

The literature review was carried out according to PRISMA guidelines. No temporal restrictions were applied. The research was performed using the follow-ing keywords: genetic cause of infertility, genetic cause of male infertility, genetic cause of female infertility, muta-tions and infertility, molecular diagnostics in infertile couples, molecular diagnostics and reproductive medi-cine, PGT techniques, and PGT and ART. All the papers found were carefully read and evaluated before their inclusion. No unpublished studies were taken into con-sideration. In addition, the following databases were also consulted to verify gene/phenotype associations: NIH (https ://www.nih.gov), OMIM (https ://www.omim.org) and OrphaNet (https ://www.orpha .net/conso r/cgi-bin/ index .php).

Results

Genetic tests in the identification of the causes of infertility It has been estimated that every healthy subject is a car-rier of 5/8 genetic alterations associated with recessive genetic disorders; therefore, even in the absence of spe-cific symptoms, family planning and reproduction can be risky. Moreover, it has been reported that almost 50% of infertility cases are related to genetic disorders [7, 8]. In the presence or high suspicion of a genetically based reproductive risk, the genetic test provides a more accu-rate diagnosis of infertility and provides the opportunity to inform the couple about the possible risk of trans-mission to the offspring. Unfortunately, genetic tests for examining infertility are based on a standard algorithm directed to investigate the most frequent genetic causes without taking into account the patient’s personal or family history. Consequently, the results are quite dis-couraging: a genetic diagnosis is reached in approxi-mately 4% of all infertile males, and approxiapproxi-mately 20%

of infertile couples remain undiagnosed. In contrast, an accurate medical and familial history aimed at identify-ing genetically based syndromes (characterized by typical dimorphisms, associated disabilities and even infertility) could direct patients to specific genetic tests [9]. Starting from this consideration, we examined the genetic disor-ders related to male and female infertility and subdivided them according to the signs and symptoms observed by the specialist during the first medical examination. Genes associated with specific and rare clinical conditions were not excluded either; they could be useful, in the context of a specific clinical picture, to request an in-depth analy-sis using a targeted genetic test. Therefore, we provide the main points on the genetic pathology, current test execu-tion modality and management of the patient (Tables 1,

2, 3, 4, 5, 6, 7).

Male genetic infertility

Genetic factors have been found in all the etiological categories of male fertility (pre-testicular, testicular and post-testicular): OMIM (Online Mendelian Inheritance in Man) reports more than 200 genetic conditions related to male infertility, ranging from the most common clini-cal presentations of infertility to the rarest complex syn-dromes in which signs and symptoms are beyond the reproductive problems [103]. In most cases, infertility is only one of the clinical signs of a complex syndrome; on the contrary, in some genetic conditions, infertility is the main phenotypic feature. Moreover, it is important to monitor these infertile patients over time because a greater morbidity and a lower life expectancy have been described for these infertile patients than for the gen-eral population and are most likely caused by the genetic abnormalities involved in male sterility [104].

Today, the presence of alterations in the spermio-gram is the first indication for genetic tests, particularly in cases of severe oligospermia (< 5 million/ml) (further parameters are hormonal levels, malformations, recur-rent abortions, and family history) [105]. Interestingly, a recent study by Oud et al. highlighted how the number of genes that are definitively linked to the more common phenotypes of oligozoospermia or azoospermia remains limited (50%); the other half are genes involved in zoospermia, although the monomorphic forms of terato-zoospermia are extremely rare [106].

Genetic disorders related to male infertility include whole chromosomal aberrations (structural or numeri-cal), partial chromosomal aberrations (i.e., microdele-tions of the Y chromosome) (listed in Tables 1, 2) and monogenic diseases (listed in Tables 3, 4, 5). In particular, abnormalities in sex chromosomes have a greater impact on spermatogenesis, while mutations affecting autosomes

Table 1 T he chr omosome ab err ations r ela ted t o t

esticular male inf

er tilit y: fr om the first obser va tion t o the r ep or t Da tabase sour

ces: NIH, OMIM and Or

phaNet AZF , az oosper mia fac tor ; ✓ , y es;

✗, no; NA, not applicable; donor

, het

er

ologous f

er

tiliza

tion with sper

m donor ; ST S, sequenc e tagged sit es Indica tions for genetic t est G enetic c ondition Fr equenc y Test Chr omosome/ genetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. A zoosper mia/oligo -zoosper mia; S er toli cell syndr ome type I and t ype II (pr esence of some

tubules with nor

mal sper mat ogenesis) and h yposper mat o-genesis diag nosis by hist olog ical evaluation M icr odeletion Y chr o-mosome AZF c 1/2.500; ( AZF c 60%, AZF b 15%, AZF b-c 22%, AZF a 3%); 13% of az oosper mia cases; 3–7% of oligo zoosper mia cases M olecular diag nosis by PCR of ST S sequences Int

erstitial deletion of AZF

c Y r eg ion (recombination bet w een palin -dr omes b2 and b4); D AZ, BPY2, PR Y2, CD Y1 ✓ : t esticu -lar sper m retr ie val + ICSI Y link ed ✓ O

ther causes of azoosper

mia or oligo zoosper mia [ 10 ] A zoosper mia; sper -mat ogenesis ar rest by hist olog ical evaluation M icr odeletion Y chr o-mosome AZF b Int

erstitial deletion of AZF

b

Y r

eg

ion

(deletions P5/ proximal-P1); RBMY

, CD Y, HSFY , PR Y A zoosper mia M icr odeletion Y chr o-mosome AZF b-c

Combined deletion AZF

b + AZF c (P5/ distal-P1 or P4/ distal-P1) ✓ : donor NA NA A zoosper mia; S er toli cell syndr ome type I diag nosis b y hist olog ical e valua -tion (i.e ., complet e absence of ger m cells in seminif er ous tubules) M icr odeletion Y chr o-mosome AZF a D eletion of AZF a Y (r ecombina -tion bet w een HER V15y q1 and HER V15y q2)

Table 2 T he chr omosome ab err ations r ela ted t o pr et

esticular male inf

er tilit y: fr om the first obser va tion t o the r ep or t Indica tions for genetic t est G enetic c ondition Fr equenc y Test Chr omosome/ genetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. H yper gonadotr opic hypogonadism, ↑FSH ↑ LH ↓ T, az oosper mia, oligo zoosper mia; small t est es , inf er -tilit y, gynecomas -tia; neur ocog nitiv e

deficits; metabolic syndr

ome , t ype 2 diabet es . Appr oxi -mat ely 10% of these subjec ts ha ve sper mat oz oa in the ejaculat e,

and in 30–50% of cases ther

e is intrat esticular sper mat ogenesis Klinef elt er ’s syn -dr ome 1/660 ne w -bor ns; > 5% in se ver e oligo zoo -sper mia; 10% in az oosper mia Kar yot ype 47,X XY (85–90%) 46,XY/47,X XY mosaicism (6–7%) 46,X X/47,X XY or multiple X ane -uploidy (3–8%) ✓ T esticular sper m retr ie val + ICSI D e no vo mutation NA 46,X X t esticular DSD [ 11 , 12 ] Shor t statur e; gyne

-comastia, male exter

nal genitalia, small t est es , cr yp -tor chidism, h ypo -spadias , inf er tilit y, ↑ FSH ↑ LH ↓ T; az oosper mia/oli -go zoosper mia Nonsyndr omic 46,X X T esticular Disor ders of S ex D ev elopment (D e la Chapelle syndr ome) 1/20.000; 0,9% in az oosper mia; 1–3% nor -mosper mia FISH or CM A SRY + X X (80–90%) ✗ T esticular sper m retr ie val; ✓ het er ologous fer tilization AD ✓ Syndr omic f or ms of 46,X X t esticular DSD; 45X/46,XY ; 47,X XY ; 46,X X; sex chr omosome

mosaicisms; Prenatal exposur

e of 46,X X f etuses t o andr ogens [ 13 ] Penoscr otal h ypo -spadias , cr ypt or -chidism, inf er tilit y; ↑ FSH ↑ LH ↓ T; az oosper mia/oli -go zoosper mia SRY − X X (< 10%) Unk no wn ✓ Shor t statur e; small test es , inf er tilit y; ↑ FSH ↑ LH ↓ T; az oosper mia/oli -go zoosper mia CM

A or molecular _diagnostic b

y PCR CNV or r ear range -ments in SO X9 , SO X3, RSPO1 and WNT4 (rar e) ✓ AD f or SO X9; AR f or RSPO1 or WNT4 ✓ 46,X X; 46,XY disor ders of sex de velopment [ 14 – 18 ]

Da

tabase sour

ces: NIH, OMIM and Or

phaNet

✓

, y

es;

✗, no; NA, not applicable; ICSI, in

tr ac yt oplasmic sper m injec tion; IVF , in vitr o f er tiliza

tion; FISH, fluor

esc enc e in situ h ybr idiza tion; PGT : pr eimplan ta tion genetic t esting; CM A, chr omosomal micr oar ra y analy sis Table 2 (c on tinued) Indica tions for genetic t est G enetic c ondition Fr equenc y Test Chr omosome/ genetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. Tall statur e, dela yed de velopment of

speech, language or mot

or sk ills , autism spec -trum disor der , hypot onia, mot or tics , clinodac tyly , scoliosis , att ention deficit h yperac -tivit y disor der ; ↑ FSH nor mal or ↓ T; fr om nor mal to az oosper mia; from 0.57 t o 77.8% sper m mosai -cism, a- or h yper diploidy D ouble Y syndr ome (Jacobs syn -dr ome) 1/1.000; 0.4% in oligo zoosper mia Cyt ogenetics t ests 47,XY Y; 46,XY/47,XY Y mosaics ✓

IVF or ICSI in case of oligosper

mic patients D oes not ha ve a clear patt er n of inher itance ✓ 46,XY [ 14 , 19 ] Subf er tilit y or une ventful andr o-log ical hist or y; oligo zoosper mia Balanced struc tural chr omosome aber rations 5% of inf er tile men FISH t(SR Y; X); der(13, 14); der(14, 21); der(14, 15) ✓ NA ✓ PGT O

ther causes of oligo

zoosper

mia

[

20

Table 3 T he genetic c auses r ela ted t o pr et

esticular male inf

er tilit y: fr om the first obser va tion t o the r ep or t M ain indica tions for genetic t est H ypogonadotr opic h ypogonadism ( CHH) O ther indica tions for genetic t est G enetic disor der Fr equenc y G enetic t est G enetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. Lack of puber ty ; micr openis , cr ypt or chid -ism; pr epuber tal testicular v olume , absence of second -ar y sexual f eatur es , decr eased muscle mass , diminished libido , er ec tile dys -func tion, inf er tilit y, lo w t est ost er one , estradiol Kallmann syndr ome (olfac togenital syndr ome with ano - or h yposmia az oosper mia) Pr evalence: 1/30,000; incidence: 1/8000 M olecular diag nosis ANOS1 ✓ X-link ed ✓ Syndr omes associat ed with hypogonadotr opic hypogonadism [ 21 , 22 ] CHD7, FGFR1, FGF8, SO X10 AD FEZF1, PR OK2, PR OKR2 AR Obesit y, r etinitis pig ment osa, post -axial poly dac tyly , kidne y dysfunc tion, beha vioral dysfunc -tion; inf er tilit y Bar det –Biedl syndr ome (Lau -rence –M oon–Biedl syndr ome) 1:100,000 Nor th Amer ica; 1:160,000 Switz er land; 1:17,500 Ne w

-foundland; 1:13,500 Bedouin, Kuwait

M ultigene panel Fr om BBS1 t o BBS19 ✓ AR ✓ M cKusick –K aufman syndr ome (MKS) [ 21 , 23 – 25 ] A dr enal insufficienc y; cr ypt or chidism, dela yed puber ty , inf er tilit y X-link ed adr enal hypoplasia con -genita 1:12,500 M olecular diag nosis NR0B1 ✓ X-link ed r ecessiv e patt er n ✓ 21-h ydr ox ylase defi -cienc y; 11-h ydr ox y-lase deficienc y [ 26 ] Diabet es mellitus , hypoth yr oidism, alopecia t otalis , long , tr iangular face , h yper telor ism; dyst onias , dysar -thr ia, dysphag ia; inf er tilit y W oodhouse –Sak ati syndr ome (diabe -tes-h ypogonadism-deafness-int el -lec tual disabilit y syndr ome) Unk no wn M olecular diag nosis DCAF17 ✓ AR ✓ Per rault syndr ome; D eafness and her editar y hear ing loss; G onadotr opin-releasing hor mone deficienc y [ 27 ] A dult -onset neur odegen -erativ e disor der ; hypogonadotr opic hypogonadism G or don Holmes syndr ome ( cer

-ebellar ataxia and hypogonadotr

opic hypogonadism) Unk no wn M olecular diag nosis RNF216, PNPLA6 ✓ AR ✓ Cer ebellar ataxia [ 28 – 30 ]

Table 3 (c on tinued) M ain indica tions for genetic t est H ypogonadotr opic h ypogonadism ( CHH) O ther indica tions for genetic t est G enetic disor der Fr equenc y G enetic t est G enetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. Cir rhosis , diabet es , car diom yopath y, ar thr itis , sk in hyper pig menta -tion; ele vat ed serum transf er rin-iron saturation ( TS); ele vat ed serum fer ritin concentra -tion; inf er tilit y Hemochr omat osis (Hemochr omat osis Type 1, HFE-A ssoci -at ed Hemochr oma -tosis , HFE-HH) 2–5:1000 nor ther n Eur opean ancestr y;

1:200–400 non- Hispanic whit

es , Nor th Amer ica G ene -tar get ed or molecular diag -nosis HFE (t ypically p.C ys282T yr and p.H is63A sp can be per for med first) ✓ AR NA Rar er pr imar y ir on ov er load disor -ders and second -ar y ir on o ver load disor ders [ 31 – 34 ] A zoosper mia/ oligo zoosper mia; ↑ LH, nor mal T , hyperandr ogenism;

feminization of the exter

nal genitalia at bir th, abnor mal secondar y sexual de velopment in puber ty , and inf er tilit y Andr ogen insensitiv -ity syndr ome ( AIS) 2–5:100,000 Scr eening f or AR mutations (> 300) AR D onor X-link ed r ecessiv e NA MRKH syndr ome; H ypospadias; M AIS; Under masculiniza -tion of ex ter nal

genitalia and puber

tal under vir i-lization [ 35 , 36 ] Glucocor ticoid and mineralocor ticoid deficiencies; h ypo -spadias; ambigu

-ous genitalia, infer

tilit y 3-β-h ydr ox yst er oid deh ydr ogenase (HSD) deficienc y Unk no wn M olecular diag nosis HSD3B2 D onor AR NA Ambiguous genitalia [ 37 ]

Table 3 (c on tinued) M ain indica tions for genetic t est H ypogonadotr opic h ypogonadism ( CHH) O ther indica tions for genetic t est G enetic disor der Fr equenc y G enetic t est G enetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. D eficiencies in GH,T SH, LH, FSH, PrL, and A C TH; hypoth yr oid

-ism; neonatal hypogly

cemia; micr openis without hypospadias , with or without cr yp -tor chidism; shor t statur e and dela yed

bone maturation; absent/dela

yed/ incomplet e secondar y sexual de velopment, inf er tilit y PR OP1-r elat ed com -bined pituitar y hor -mone deficienc y

1:4000 in England and the US

M olecular diag nosis PR OP1 ✓ AR ✓ CPHD; isolat ed gr owth hor mone deficienc y; isolat ed hypogonadotr opic hypogonadism [ 38 , 39 ] Ambiguous genitalia or ex ter nal geni

-talia that appear female; micr

openis

and h

ypospadias;

not much facial or body hair

; inf er tilit y 5-Alpha r educ tase deficienc y (familial incomplet e male pseudoher maphr o-ditism, t ype 2) Unk no wn M olecular diag nosis SRD5A2 ✓ AR ✓ Ambiguous genitalia [ 40 – 42 ] Da tabase sour

ces: NIH, OMIM and Or

phaNet

✓

, y

es;

✗, no; NA, not applicable; donor

, het

er

ologous f

er

tiliza

tion with sper

Table 4 T he genetic c auses r ela ted t o t

esticular male inf

er tilit y: fr om the first obser va tion t o the r ep or t Indica tions for genetic t est G enetic disor der Fr equenc y G enetic t est Chr omosome/ genetic alter ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. M aldescended t est es

Absence of one or both test

es fr om the scr otum; nonobstruc tiv e az oosper mia; hypogonado -tr opic h ypog -onadism Cr ypt or chidism 2%; 20% of inf er tile men; 30/80% of azoosper mia M olecular diag -nosis INSL3; L GR8 ✓ AD ✓ H ypogonadotr opic

hypogonadism; Noonan and Prader

–W illi syndr ome [ 43 , 44 ] H yper tension, hypok alemic alk alosis; lack of secondar y sex -ual charac ter -istics; t esticular feminization 17 alpha(α)- hy dr ox ylase/17,20-lyase deficienc y 1 in 1 million M olecular diag -nosis CYP17A1 D onor AR NA Ambiguous geni -talia [ 45 ] S ev er e muscular hypot onia, geni -tal h ypoplasia, incomplet e puber tal de vel -opment, inf er til -ity ; cr ypt or -chidism (93%); obesit y, mental retar dation (lat e onset) Prader –W illi syndr ome (P W S, Prader –Labhar t– W illi syndr ome) 1:10,000 t o 1:30,000 DNA meth -ylation testing; Cyt ogenetic/ FISH/chr omo -somal micr oar -ra y findings: deletion in bands 15q11.2-q13 (70%) 15q11.2 r eg ion D onor Pat er nal deletion; mat er nal unipa -rental disom y15 ✓ Cr ypt or chidism; Craniophar yn -gioma [ 21 , 46 – 48 ] Shor t stat -ur e, facial dysmor phism, congenital hear t def ec ts , sk eletal def ec ts , w ebbed neck , mental r etar da

-tion, bleeding diathesis; ear

ly onset Noonan syndr ome -1 (NS1) 1:1000–2500 G

ene sequencing star

ting with

PTPN11, f

ollo

w

ed

by SOS1, KRAS and RAF1

PTPN11

(>

50%),

SOS1 (10–15%), KRAS (5%), RAF1 (3–17%)

✓ AD ✓ Tur ner syndr ome; cr ypt or chidism; az oosper mia [ 49 ]

Table 4 (c on tinued) Indica tions for genetic t est G enetic disor der Fr equenc y G enetic t est Chr omosome/ genetic alter ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. G onadal dysgen -esis , ambiguous genitalia, inf er -tilit y; incr eased risk of W ilms tumor D en ys–Drash syn -dr ome Unk no wn M olecular diag -nosis W T1 – AD ✓ Frasier syndr ome [ 50 , 51 ] A tr oph y of the abdominal muscles , malf or

-mations of the urinar

y trac t Prune –belly syn -dr ome ( other names Syndr om of Eagle –Bar ret; syn -dr om of Obr insk y) 1/35,000 and 1/50,000 bir ths

and 95% of cases occur in males

M olecular diag -nosis CHRM3 NA – NA M egac ystis/ megaur et er or post er ior ur ethral valv es [ 52 , 53 ] Ost eopor osis; hyper gly cemia; ambiguous genitalia Ar omatase deficienc y Unk no wn M olecular diag -nosis CYP19A1 ✓ AR ✓ O

ther condition of estr

ogen deficienc y [ 54 , 55 ] P ropor tionat e shor t statur e, dela yed closur e of f ontanelles , pr ominent for ehead , dr ooping shoul -ders , abnor mal dental de velop -ment; ear ly onset Cleidocranial dys -plasia 1:1,000,000 M olecular diag -nosis RUNX2 ( CBF A1) ✓ AD; de no vo

pathogenic variant

✓ Py cnodysost osis; mandibuloa -cral dysplasia; CBFB [ 21 , 56 ] Syndr

omic without maldescended t

est es Shor t statur e, telang iec tatic er ythemat ous sk in lesions , high r isk f or malig nancies; ear ly onset; az oosper mia or se ver e oligosper mia Bloom ’ s syndr ome (Bloom– Tor re – M achacek syn -dr ome) Rar e disor der M olecular diag -nosis BLM ✓ AR ✓ REC Q -mediat ed genome instabil -ity ; A taxia–t el -ang iec tasia;

Fanconi; anemia; Nijmegen br

eak -age syndr ome; W er ner syn -dr ome [ 21 , 57 ]

Table 4 (c on tinued) Indica tions for genetic t est G enetic disor der Fr equenc y G enetic t est Chr omosome/ genetic alter ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. Shor t statur e, macr ocephaly , distinc tiv e face (small , tr ian

-gular face with prominent f

or e-head , nar ro w chin, small ja w), dela yed de vel

-opment, speech and language problems

, lear

n-ing disabilities; digestiv

e sys -tem abnor mali -ties; micr openis; ear ly onset Russel–Silv er syn -dr ome Pr evalence: unk no wn; esti -mat ed incidence ranges fr om 1 in 30,000–1 in 100,000 people M eth ylation M eth ylation in volv

-ing H19 and IGF2

✓

Sporadic; unipa

-rental disom

y

Usually not pos

-sible Intraut er ine g ro wth retar dation and shor t statur e [ 21 , 58 , 59 ] K erat oconus ,

glaucoma, and myopia as w

ell

as fr

om malf

or

-mations of the brain, sk

elet on, and k idne y; impair ment of r espirat or y func tions; inf er tilit y (asthe -no zoosper mia and abnor mal flagellar mor -phology) Pr imar y ciliar y dysk i-nesia (PCD) Pr evalence: 1:16,000; 1:400 in a V olendam population r esid

-ing in a fish-ing village of Nor

th Holland M olecular diag -nosis

DNAH5 (30%), DNAI1 (10%) and T

XNDC3,

DNAH11, DNAI2 (rar

e); 60% gene loci unk no wn ICSI AR ✓ Chr onic sinopul -monar y disease and br onchiec -tasis [ 21 , 60 ]

Table 4 (c on tinued) Indica tions for genetic t est G enetic disor der Fr equenc y G enetic t est Chr omosome/ genetic alter ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. M ultisyst em disor der aff ec t-ing the sk eletal

and smooth muscles

, the hear t, the ey es , and the endocr ine and central ner vous syst ems . M ental retar dation; inf er tilit y M yot onic dystr oph y 1 (M or bus C ur -schmann–St einer t, D ystr ophia m yo -tonica 1, DM1) 1 in 8000 M olecular diag -nosis of the C TG

repeat expansion in the DMPK gene (>

50 C TG repeats r esult in DM1) DMPK ✓ AD ✓ Prader –W illi syndr ome , nemaline m yo -path y, X -link ed centr onuclear m yopath y; DM2; Her editar y distal m yopathies; Her editar y m yo -tonia [ 21 , 61 ] Bone mar ro w failur e, h ypopig -mentation, shor t statur e, ph ysical abnor malities , or gan def ec ts (gastr oint estinal abnor malities; hear t def ec ts; and ey e abnor mali -ties , malf or med

ears and hear

ing

loss); incr

eased

risk of cer

tain

cancers; and malf

or mations of the r epr oduc -tiv e syst em and inf er tilit y Fanconi anemia 1 in 160,000 (mor e common among people of Ashk enazi J ewish

descent, the Roma popula

-tion of Spain, and black S

outh A fricans) M olecular diag -nosis FANCA, F ANC C and FANC G (90%) NA

AR; AD:RAD51- relat

ed F A; X-link ed: F ANCB-relat ed F A ✓ Bloom syndr ome; ataxia–t elan -giec tasia; NBS; Seck el syndr ome; neur ofibr oma -tosis 1 [ 62 , 63 ] Nonsyndr omic inf er tilit y Abnor mal sper m cells (r ound

head and no acrosome) and infer

tilit y Globo zoosper mia (sper mat ogenic failur e 9) Rar e (1:65,000); common in North A frica:

1:100 cases of male inf

er tilit y M olecular diag no

-sis of DPY19L2, follo

w ed b y SP AT A16 DPY19L2 homo zy

-gous deletion, point mutations; SPAT

A16 ✓ ICSI + A OA AR ✓ Sper mat ogenic failur e [ 64 , 65 ]

Table 4 (c on tinued) Indica tions for genetic t est G enetic disor der Fr equenc y G enetic t est Chr omosome/ genetic alter ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. Abnor mal sper m cells (abnor -mally lar ge and misshapen heads , contains ex tra chr omo

-somes; multiple flagella, most often f

our) and inf er tilit y M acr oz oosper mia (sper mat ogenic failur e 5) Unk no wn;1:10,000 males in Nor th A frica M olecular diag -nosis AURK C mutations (c .144delC, 85%; p.Y248, DR 13%) D onor AR NA Sper mat ogenic failur e [ 66 – 68 ] P rimar y inf er til -ity ; multiple mor pholog ical abnor malities of sper m flagella (absent, shor t, coiled , bent, and ir regular flagella); asthe -no zoosper mia M ultiple mor pholog i-cal abnor malities of the sper m flagella (sper mat ogenic failur e 18) Unk no wn M olecular diag -nosis DNAH1 mutation (c .8626-1G > A; c.3860 T > G) ✓ ICSI AR NA Ciliar y dysk inesia pr imar y [ 69 ] G enital abnor mal -ities; h ypoplasia of L ey dig cells; micr openis , hypospadias , bifid scr otum, ambiguous genitalia Le ydig cell h ypo -plasia (h yper -gonadotr opic hypogonadism due to LHC GR def ec t) Unk no wn M olecular diag -nosis LHC GR D onor AR ✓ H yper gonado -tr opic h ypog -onadism [ 70 , 71 ] A stheno -zoosper mia; absence of an y other symp -toms CA TSPER-r elat ed nonsyndr omic male inf er tilit y Unk no wn M olecular diag -nosis CA TSPER1, GALNTL5 D onor AR ✓ M ale inf er tilit y [ 69 , 72 , 73 ]

Table 4 (c on tinued) Indica tions for genetic t est G enetic disor der Fr equenc y G enetic t est Chr omosome/ genetic alter ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. Nor mal general ph ysical exami

-nation, absence of clinical find

-ings in volving other or gan syst ems; t ypical female ex ter nal genitalia, ut erus

and fallopian tubes nor

mally for med , gonadal dys -genesis; sk eletal abnor malities , campomelic dysplasia Sw yer syndr ome (46,XY complet e gonadal dysgen -esis) 1 in 80,000 M olecular diag -nosis SRY (15%); M AP3K1

(18%); DHH and NR5A1 (rar

e) AR T D e no vo; rar e AD ✓ Ambiguous geni

-talia and/or sex chr

omosome -phenot ype discor dance [ 13 ] A stheno zoo -sper mia; hear -ing loss D eafness-inf er tilit y syndr ome (DIS) Unk no wn CM A/ar ra y-CGH Homo zy gous dele -tion at 15q15.3 including CA T-SPER2, STR C D onor AR ✓ DFNB16 [ 74 , 75 ] Nonobstruc tiv e az oosper mia Small t est es and inf er tilit y, with se ver e oligo -zoosper mia or nonobstruc tiv e az oosper mia due t o matura -tion ar rest at the pr imar y sper mat oc yt e stage M eiotic ar rest at pr i-mar y sper mat oc yt e stage (sper mat o-genic failur e 25) Unk no wn M olecular diag -nosis TEX11 D onor X-link ed NA Sper mat ogenic failur e [ 76 – 78 ]

Table 4 (c on tinued) Indica tions for genetic t est G enetic disor der Fr equenc y G enetic t est Chr omosome/ genetic alter ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. Nonobstruc tiv e az oosper mia, inf er tilit y,

testicular biopsy sho

wing

absence of sper

mat

ogenic

cells and a Ser

toli cell-only patt er n Sper mat ogenic failur e 32 Unk no wn M olecular diag -nosis SOHLH1 D onor AD NA Sper mat ogenic failur e [ 79 ] A zoosper mia; t es -ticular hist ology sho wing ar rest of sper mat

o-genesis at the pach

yt ene stage of pr imar y sper mat oc yt es Sper mat ogenic failur e 4 (SPGF4) 1% M olecular diag -nosis SY CP3 ( COR1 RPR GL4 SCP3 SPGF4) D onor AD NA Sper mat ogenic failur e [ 80 ] A zoosper mia or oligo zoo -sper mia Sper mat ogenic failur e, Y -link ed 2 Unk no wn M olecular diag -nosis RBMY1A1, D AZ1–4 D onor Y-link ed NA Sper mat ogenic failur e [ 81 , 82 ] Da tabase sour

ces: NIH, OMIM and Or

phaNet

✓

, y

es;

✗, no; NA, not applicable; donor

, het

er

ologous f

er

tiliza

tion with sper

m donor ; A O A, assist ed o var ian ac tiv ation, CM A, chr omosomal micr oar ra y analy sis

Table 5 T he genetic c auses r ela ted t o p ostt

esticular male inf

er tilit y: fr om the first obser va tion t o the r ep or t Da tabase sour

ces: NIH, OMIM and Or

phaNet

✓

, y

es;

✗, no; NA, not applicable; ICSI, in

tr ac yt oplasmic sper m injec tion M ain indica tions for genetic t est Obstruc tiv e az oospermia or se ver e oligospermia AR T Inheritanc e A nt ena tal test D iff er en tial diag nosis Re fs. G enetic disor der Fr equenc y G enetic t est G enetic alt er ation Abnor malities of seminal

vesicles or absence of vas def

er ens; nor mal testicular de velop

-ment and func

tion; nor mal sper mat ogen -esis; a lo w v olume of ejaculat ed semen with a specific pr ofile (v olume < 1.5 ml , ph < 7.0, ele vat ed citr ic

acid concentration, elevat

ed acid phos

-phatase concentration, low fruc

tose concen

-tration, and failur

e t o coagulat e) Congenital bilat eral

absence of the vas def

er ens ( CBA VD) 25%; 1–2% in inf er tilit y Scr eening f or CFTR mutations Tw o CFTR pathogenic var iants identified (46%); one CFTR path -ogenic var iant identi -fied (79%) ✓ ICSI AR ✓ Young syndr ome; Her editar y ur o-genital dysplasia [ 83 – 85 ] M ultisyst em disease aff ec ting epithelia of the r espirat or y trac t, ex ocr ine pancr eas , int estine , hepat obiliar y syst em, and ex ocr ine sw

eat glands; obstruc

-tiv e az oosper mia and male inf er tilit y Cystic fibr osis 1:3200; CF occurs with lo w er fr equenc y in

other ethnic and racial populations (1:15,000 African Amer

icans , and 1:31,000 A sian Amer icans) Scr eening f or CFTR mutations Tw o CFTR pathogenic var iants identified ✓ ICSI AR ✓ A

sthma; congenital airwa

y anoma -lies; pr imar y ciliar y dysk inesia; Sh wachman–Dia -mond syndr ome; Br onchiec tasis

with or without elevat

ed sw eat chlor ide; Isolat ed hyper chlor hidr o-sis; C ongenital bilat eral absence

of the vas def

er -ens ( CBA VD) [ 84 , 86 ]

Table 6 T he genetic c auses r ela ted t o o varian f emale inf er tilit y: fr om the first obser va tion t o the r ep or t Indica tions for genetic t est G enetic _disor der Fr equenc y G enetic t est Chr omosome/genetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. POI Shor t statur e, sk eletal abnor -malities , k idne y pr oblems , w ebbed neck , lymphedema; ovar ian hypofunc tion or pr ematur e ovar ian failur e, inf er tilit y Tur

ner (45,X) (other names monosom

y X, T S) 1 in 2500 Kar yot ype M onosom y X: 45,X0 ✓ -donor Not inher ited NA POF [ 87 ] A sympt omatic

(only 10% of individuals with trisom

y X ar

e

ac

tually diag

-nosed); tall stat

-ur e, epicanthal folds , h ypot onia and clinodac -tyly ; r enal and genit our inar y abnor malities; psy cholog ical pr oblems Tr isom y X 1/1000 Kar yot ype 47X XX or mosaic ✓ NA ✓ Irr

egular menstrual cycles

, ear ly menopause , pr ematur e ovar ian failur e, inf er tilit y Frag ile X-associat ed pr imar y o var ian insufficienc y (pr ematur e ovar ian failur e 1)

1 in 200 (4/6% of all cases of POI)

M olecular diag -nosis of pr emu

-tations in the FMR1 gene on chr

omosome Xq27.3 ( CGG seg ment is repeat ed 55 t o 200 times) FMR1 gene ✓ -donor X -link ed ✓ POF [ 88 ] H ypogon -adotr opic hypogonadism; hypot onia, poor feeding , v omit -ing , w eight loss , jaundice; impair ed gr owth, cog ni -tiv e deficit and catarac ts G alac to -semia (galac -tose -1-phosphat e ur idyltrans -ferase defi -cienc y) pr evalence unk no wn; incidence 1/40,000– 60,000 M olecular diag -nosis G AL T, G ALK1 , and G ALE genes (9p13, 17q24, 1p36) ✓ AR ✓ POF

Table 6 (c on tinued) Indica tions for genetic t est G enetic _disor der Fr equenc y G enetic t est Chr omosome/genetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. Chr onic _mucocutane -ous candidiasis , hypoparath

y-roidism and aut

oimmune adr enal failur e; ear ly onset A ut oimmune poly glandular syndr ome (t ypes 1) Pr evalence: 1–9 in 1,000,000; 1/25,000 in Finland M olecular diag -nosis AIRE gene (21q22.3) ✓ AR ✓ IPEX syndr ome; aut oimmune poly endo -cr inopath y type 2 H yper tension, hypok alemia; abnor mal sexual de velop -ment, amenor -rhea, inf er tilit y 17α-h ydr ox ylase deficienc y 1 in 1 million M olecular diag -nosis CYP17A1 gene D onor AR NA Se ver e con -genital adr enal hyper plasias [ 45 ] M

ineralization of bones and osteopor

osis; hyper gly cemia; ambiguous genitalia, o var -ian c ysts ear ly in childhood , ano vulation; hirsutism Ar omatase defi -cienc y unk no wn M olecular diag -nosis CY P19A1 gene D onor AR NA PC OS [ 62 , 63 ] Ophthalmic disor -der associat ed with pr ematur e ovar ian failur e; ear ly onset Blephar ophi -mosis , pt osis ,

epicanthus inversus syn

-dr ome t ype I (BPES, t ype I) Pr ev a-lence: 1–9/100 000 M olecular diag -nosis FO XL2 gene ✓ AD or de no vo ✓ PC OS [ 89 ] Pr e- and postna -tal g ro wth r etar

-dation, facial sun-sensitiv

e telang iec tatic er ythema, incr eased susceptibilit y t o inf ec tions , and pr edisposition to cancer Bloom syndr ome Unk no wn; 1/48,000 among people of A shk enazi Je wish descent Cyt

ogenetic or molecular diag

-nosis 15q26.1; BLM gene ✓ AR ✓ Silv er –Russell syndr ome ,

Rothmund– Thomson syn

-dr ome , ataxia– telang iec tasia, Cock ayne syndr ome , and N ijmegen br eak age syndr ome

Table 6 (c on tinued) Indica tions for genetic t est G enetic _disor der Fr equenc y G enetic t est Chr omosome/genetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. O vulation disor ders (not POI) H yper gon -adotr opic amenor rhea; lack of puber ty ; absence of secondar y sexual f eatur es , decr eased muscle mass , diminished libido , inf er tilit y Kallmann pr evalence: 1/30,000; inci -dence: 1/8,000 M olecular diag -nosis

Type 1: ANOS1 Type 2 and 6: CHD7, FGFR1, FGF8 and SO

X10 Type 3: FEZF1, PR OK2, PR OKR2 ✓ X-link ed AD AR ✓ Syndr omes associat ed with hypogonado -tr opic h ypog -onadism Diabet es mellitus , hypoth yr oid

-ism, alopecia totalis

, long , tr iangular face , hyper telor ism; dyst onias , dysar thr ia, dysphag ia W oodhouse – Sak ati syn -dr ome Unk no wn M olecular diag -nosis DCAF17 gene D onor AR NA Diabet es; h ypo

-gonadism; deafness- intellec

tual disabilit y [ 27 ] Hear ing loss; int ellec tual disabilit y, ataxia, per ipheral neur opath y; ovar ian dysgen -esis , pr imar y amenor rhea, pr imar y o var ian insufficienc y, nor mal ex ter nal genitalia, infer tilit y Per rault syn -dr ome Ra re M olecular diag -nosis T WNK ; CLPP ; HARS; LARS2; HSD17B4 D onor AR NA G

onadal dysgenesis; sensor

ineural deafness [ 90 ] G onadal dysgen -esis , X X t ype , with deafness O var ian dys

-genesis with sensor

ineural

Table 6 (c on tinued) Indica tions for genetic t est G enetic _disor der Fr equenc y G enetic t est Chr omosome/genetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. Pr imar y amenor -rhea, inf er tilit y, poly cystic o var -ian syndr ome ,

hirsutism, ambiguous genitalia

Cyt ochr ome P450 o xi -dor educ tase deficienc y Unk no wn M olecular diag -nosis POR gene D onor AR NA PC OS [ 91 ] Sk eletal abnor malities , craniosynost o-sis , a flatt ened mid-face , a pr ominent for ehead , and lo w-set ears; arachnodac -tyly , choanal atr esia; pr imar y amenor rhea, inf er tilit y, poly cystic o var -ian syndr ome ,

hirsutism, ambiguous genitalia

Antle y–Bixler syn -dr ome Unk no wn M olecular diag -nosis FGFR2 gene D onor AR NA PC OS [ 92 ] Obesit y, hir

-sutism, and amenor

rhea ar e clinical cor relat es of enlar ged poly -cystic o var ies Poly cystic o var y syndr ome (PC OS) 6 t o 10% of women w or ld -wide M olecular diag -nosis A OPEP ; AR; DENND1A; ERBB4; FSHB; FSHR; FT O; GA

TA4; HMGA2; INSR;

KRR1; LHC

GR; RAB5B;

RAD50; SUMO1P1; SUO

X; THAD A; TO X3; Y AP1 ✓ D oes not ha ve a clear patt er n of inher itance NA Amenor rhea [ 93 , 94 ] Poly cystic o var y syndr ome

1 (STEIN- LEVENTHAL SYNDR

OME HYPERANDR O -GENEMIA) M olecular diag -nosis PC OS1 ✓ AD NA Amenor rhea; HYPERANDR O -GENEMIA [ 93 , 94 ]

Table 6 (c on tinued) Indica tions for genetic t est G enetic _disor der Fr equenc y G enetic t est Chr omosome/genetic alt er ations AR T Inheritanc e A nt ena tal t est D iff er en tial diag nosis Re fs. H ydr opic

placental villi, trophoblastic hyper

plasia, and poor f etal de velopment Recur rent h yda -tidif or m mole

-type 1 (familial recur

rent hy datidif or m mole , FRHM) 1:250 in east er n A sia M olecular diag -nosis NLRP7 gene (55%); KHDC3L gene (5%) ✓ AR ✓ H ydatidif or m mole [ 95 , 96 ] Abnor mally de veloped embr yo and

placenta that result in the formation of hydatidif

or m moles H ydatidif or m mole 1:1500 in USA M olecular diag -nosis C11 or F80, MEI1, REC114 ✓ AR ✓ FRHM [ 95 , 96 ] Nor mal general ph ysical exami

-nation, absence of clinical find

-ings in volving other or gan syst ems; t ypical female ex ter nal genitalia, nor -mally f or med ut erus and fallopian tubes , gonadal dys -genesis; sk eletal abnor malities , campomelic dysplasia Sw yer syndr ome (46,XY com -plet e gonadal dysgenesis) 1 in 80,000 M olecular diag -nosis SRY (15%); M AP3K1 (18%);

DHH and NR5A1 (rar

e) AR T D e no vo; rar e AD ✓ Ambiguous geni

-talia and/or sex chr

omosome -phenot ype discor dance [ 69 ] Da tabase sour

ces: NIH, OMIM and Or

phaNet

✓

, y

es;

✗, no; NA, not applicable; POF

, pr ema tur e o var ian failur e; PC OS, poly cy stic o var ian syndr ome

Table 7 T he genetic c auses r ela ted t o p ost ov arian f emale inf er tilit y: fr om the first obser va tion t o the r ep or t Da tabase sour

ces: NIH, OMIM and Or

phaNet

✓

, y

es;

✗, no; NA, not applicable; POI, pr

imar y o var ian insufficienc y Indica tions f or genetic test G enetic disor der Fr equenc y G enetic t est G enetic alt er ations AR T Inheritanc e A nt ena tal test D iff er en tial diag nosis Re fs. Under de veloped or absent ut erus and abnor malities of other repr oduc tiv e or gans; nor mal f emale ex ter nal genitalia, br easts; hyperandr ogen

-ism; facial hirsut-ism; primar

y amenor rhea; inf er tilit y M üller

ian aplasia and

hyperandr

ogenism

(other names: Biason– Lauber syndr

ome , WNT4 deficienc y) Ra re M olecular diag nosis WNT4 gene NA AD or de no vo ✓ Abnor malities of the repr oduc tiv e syst em [ 97 – 99 ] Vag ina and ut erus t o be under de veloped

or absent, although exter

nal genitalia ar e nor mal , pr imar y amenor rhea M ay er –R ok itansk y– Küst er –Hauser (MRKH) syndr ome (t ype 1) 1 in 4500 M olecular diag nosis ESR1, O XTR, WNT9B NA AD ✓ Abnor malities of the repr oduc tiv e syst em [ 100 – 102 ] Under de veloped or absent vag ina and ut erus , although ex ter nal genitalia ar e nor mal; pr imar y amenor rhea; unilat eral renal agenesis; sk eletal abnor malities; hear ing loss or hear t def ec ts M ay er –R ok itansk y– Küst er –Hauser (MRKH) syndr ome (t ype 2) Bone mar ro w failur e, hypopig mentation, shor t statur e, ph ysical abnor malities , or gan def ec ts (gastr oint es -tinal abnor malities; hear t def ec ts; e ye abnor malities , malf or

med ears and

hear

ing loss), and

an incr eased r isk of cer tain cancers; abnor mal genitalia or malf or mations of the repr oduc tiv e syst em and inf er tilit y Fanconi anemia (F anconi panc yt openia Fanconi panm yelopath y) 1 in 160,000 (mor e

common among people of A

shk

enazi

Je

wish descent, the

Roma population of Spain, and black S

outh A fricans) M olecular diag nosis FANCA, F ANC C and FANC G (90%) NA

AR; AD (RAD51- relat

ed F A); X -link ed (F ANCB-r elat ed F A). ✓ Bloom syndr ome; ataxia–t elang iec ta -sia, N ijmegen br eak -age syndr ome (NBS); Seck el syndr ome; neur ofibr omat osis 1; POI [ 62 , 63 ]

are more related, for example, to hypogonadism, terato-spermia or asthenozooterato-spermia and to familial forms of obstructive azoospermia.

Currently, the main genetic tests routinely used for the diagnosis of male infertility are the karyotype, the study of chromosome Y microdeletions, and the analysis of the CFTR gene. Since it has been reported that several mutated are related to male infertility, it is not surprising that in ~ 40% of all cases of male infertility, the underly-ing genetic pathogenesis remains unknown [107, 108]. It must also be considered that the role of de novo muta-tions should be further investigated, especially in light of what happens for Klinefelter syndrome and AZF deletions that occur almost exclusively de novo [106]. Therefore, to improve and personalize the entire diag-nostic–therapeutic pathway of male infertility, targeted genetic tests should be performed in the presence of spe-cific clinical pictures, always after appropriate genetic counselling: (1) for diagnostic purposes, (2) during clini-cal decision-making to establish the most appropriate ART strategy (for example, in the presence of deletions of the AZFa and AZFb regions, the possibility of sperm recovery using testicular biopsy is extremely low), and (3) for prognostic purposes (to establish the risk of transmit-ting the pathology and plan a prenatal or preimplantation diagnostic procedures).

Whole chromosomal aberrations The prevalence of

chromosomal alterations varies from 1.05 to 17% (this gap depends on the characteristics of the studied group) but is 0.84% in newborns [109]. Structural chromo-somal rearrangements are more common with respect to numerical abnormalities; this does not apply to sex chro-mosomes whose abnormalities, accounting for approxi-mately 4.2% of all whole chromosomal aberrations, are represented by sex chromosome aneuploidies in 84% of cases and by structural rearrangements of chromosome Y in the remaining 16% of cases. Klinefelter syndrome (karyotype 47, XXY) is the most frequent type of sex chromosome aneuploidy detected in infertile men [11,

12]; the second most frequent gonosomal abnormality is Double Y syndrome or Jacobs syndrome, character-ized by the presence of Y chromosome disomy [14, 110]. In addition to reduced reproductive potential, carriers of chromosomal abnormalities have an increased risk of abortion or generate a child with an abnormal karyotype. For this reason, Table 1 shows the main chromosomal aberrations that could interfere with healthy reproduc-tion, the relative information on the phenotypic aspect, the laboratory tests to highlight them and the indications for antenatal genetic testing.

Partial chromosomal aberrations Microdeletions in

the long arm of the Y chromosome (Yq), named the AZF (Azoospermia Factor) region, have been found in 8–12%

of azoospermic men and 3–7% of oligozoospermic men [106], resulting in the most common molecular genetic cause of male infertility [110]. The AZF region includes three groups of genes (AZFa, AZFb and AZFc) that are most responsible for spermatogenesis, so partial or complete deletions in this area may impair reproduc-tive capacity. Indications for AZF deletion screening are based on sperm count (< 5 × 106 _{spermatozoa/ml)} associ-ated with primary testiculopathy, and ICSI is required to overcome infertility [111].

Male offspring will carry the same father’s Yq microde-letions or even a worse one; therefore, genetic counsel-ling is mandatory [112]. Parents should be aware of the risk of having a child affected by Turner’s syndrome (45, X0) or other phenotypic anomalies associated with sex chromosome mosaicism [113].

The rearrangement of the AZFc zone is responsible for 60% of all Yq microdeletions [114]. The AZFc region (3.5 Mb) contains several copies of five repeats (b1, b2, b3, b4, and gr), whose similarity and large size predispose an individual to a relatively high incidence of de novo deletions via homologous recombination [115]. The most common is the loss of the whole b2/b4 region, which includes the DAZ family (Deleted in Azoospermia) and leads to spermatogenesis deterioration [115, 116]. More details about AZF are reported in Table 1.

Single gene mutations This section will focus on the

noteworthy single gene disorders that have clinical rel-evance for male infertility (Tables 3, 4, 5). Although thou-sands of genes are involved in male infertility, today, only a handful of genetic diseases are routinely investigated (e.g., cystic fibrosis) [117, 118]. As shown by several stud-ies, the approaches to identify a single causative gene are not useful considering that more than 2300 genes are expressed in the testis alone and that hundreds of them influence reproductive functions and can contribute to male infertility. Even if nearly 50% of infertility cases are due to single or multiple genetic defects, the genetic causes remain unexplained for 20% of patients [3]. Fur-thermore, the increasingly widespread use of tools, such as NGS (next-generation sequencing), for both diagnostic and research purposes will allow us to rapidly expand our knowledge of this field [4].

Starting from the clinical and laboratory evaluation, as shown in Tables 3, 4, 5, the main genetic conditions that could interfere with healthy reproduction are reported with the aim of improving the targeted genetic test in the presence of specific clinical pictures.

Female genetic infertility

In contrast to male infertility, little is known about the genetic bases of female infertility. Accordingly, fewer spe-cific tests are routinely recommended to infertile females

to investigate the presence of chromosomal disorders or single-gene defects related to their clinical pheno-types. Indeed, isolated infertility due to genetic causes is rare; more commonly, syndromic diseases contribute to female infertility. To date, genetic tests are mainly used for patients with POI, limited to chromosomal aberra-tions and FMR1 premutaaberra-tions. We therefore focused on the description of these two conditions; however, as shown in Tables 6, 7, more details have been reported concerning the main chromosomal and genetic altera-tions that could interfere with healthy reproduction; for each of them, the main phenotypic presentations and the laboratory tests that are available in the pre- and postna-tal periods are reported.

Whole chromosomal aberrations Considering that

chromosomal disorders significantly impact fertility and the miscarriage risk, karyotype analysis is always advisable [119]. The most clinically important struc-tural disorders in infertile females are translocations, both reciprocal (exchange of two terminal segments from different chromosomes) or Robertsonian (centric fusion of two acrocentric chromosomes) responsible for blocks of meiosis and structural alterations of the X chromosome. Patients with reciprocal translocations are at a significantly increased risk of infertility, includ-ing hypogonadotropic hypogonadism with primary or secondary amenorrhea or oligomenorrhea. The balanced rearrangements do not create health problems for their carriers because they cause neither loss nor duplication of genetic information, but they can give rise to gametes in which the genetic information is unbalanced and can thus become a cause of infertility or multiple miscarriage. Some abnormalities, such as the XXX karyotype, could not be clearly associated with infertility.

Women with a normal karyotype produce a variable percentage of oocytes with chromosomal abnormali-ties due to errors occurring during crossing-over and/or meiotic nondisjunction [120, 121]. The three main classes of abnormalities are 45X, trisomy and polyploidy. It is well known that these events increase with maternal age [122]. It is possible to analyze gametes or embryos while undergoing ART thanks to PGT. The efficacy of the tech-nique is increased after screening for aneuploid embryos and transferring only euploid embryos [123, 124].

Fragile X syndrome Fragile X syndrome is an

autoso-mal dominant genetic disorder caused by the presence of over 200 repetitions of the CGG triplet sequence in the

FMR1 (Fragile X Mental Retardation 1) gene or by a

dele-tion affecting the FMR2 (Fragile X Mental Retardadele-tion 2) gene. Carriers of the female FMR1 premutation (when the number of CGG repeats falls between 55 and 200) or FMR2 microdeletion show menstrual dysfunction,

diminished ovarian reserve, and premature ovarian fail-ure [125, 126].

In addition to the family history, in the case of women with these clinical manifestations, the possibility of a molecular test should be considered. The most common genetic contributors to POI are X-chromosome-linked defects. In rare cases, the cause is an alteration in an autosomal chromosome [88]. Identifying the mutations in a timely fashion is of paramount importance to man-aging the reproductive options and, if necessary, choos-ing a preimplantation genetic diagnosis program: the aim is to identify the specific clinical pictures in which a tar-geted genetic test could guide a personalized diagnostic– therapeutic treatment approach.

Molecular approaches in the identification of genetic diseases that are transmissible to offspring

It is well known that in 20–25% of cases, perinatal mor-tality is caused by inherited chromosomal or genetic alterations [127]. Thanks to medical awareness in recent decades, preconception carrier screening has become widely requested. The identification of couples at risk of transmitting a specific inherited disorder to their off-spring offers the possibility of making informed repro-ductive choices to future parents. If the reprorepro-ductive partner happens to carry a gene alteration for one of the genetic conditions, the pregnancy would be at risk for a child with that disease.

The American College of Obstetricians and Gynecolo-gists has issued standard recommendations for ethnic and general population genetic screening in couples based on reproductive age [128]. Testing is available for more than 2000 genetic disorders, including common diseases, such as sickle-cell anemia, cystic fibrosis, and spinal muscular atrophy, or more complex conditions, such as mental retardation and congenital heart disease.

In this context, genetic counselling is crucial for recog-nizing the genetic risk, referring patients appropriately and informing patients about genetic issues that are rel-evant to decision-making [129]. In fact, preconception carrier screening provides genetic information for mul-tiple disorders; thus, all carrier couples can make repro-ductive decisions based on their results. The tailored genetic test is a crucial tool to improve short-term and long-term outcomes for mothers and their babies [130,

131].

Currently, during the antenatal period, a variety of techniques are available to identify a transmissible disor-der to the offspring in the presence of carrier or affected couples. Each of these techniques can be applied only during a specific time period of pregnancy or at different embryo stages in the IVF protocol.

Invasive PND is usually performed on DNA extracted from fetal cells obtained by chorionic villus sampling (CVS) (between the 11th and 13th weeks of gestation) or from amniocytes (from the 15th to the 20th week), and the result is obtained in 7 or 15  days, respectively [132]. The molecular diagnosis for monogenic disease, as we detailed in a previous publication, is carried out by direct mutation analysis when the parental mutations are known or by linkage analysis when the parental muta-tions are unknown [5, 132]. Paternity verification and contamination analysis are always performed in addition to the specific analytic phases [5].

An increasing amount of interest has been shown regarding the noninvasive prenatal diagnosis (NIPD) of monogenic disease that is able to detect fetal genetic alterations in maternal blood at an early gestational age (approximately 10  weeks). However, although nonin-vasive prenatal testing (NIPT) of cell-free fetal DNA (cffDNA) for the screening of chromosomes 21, 18, 13, X and Y has been clinically adopted, NIPD remains a chal-lenge [133]. Very recently, NIPD for clinical use has been adopted in cases of sex-linked disorders and RHD [134]. Several studies have tested the application of NIPD in monogenic diseases, such as β-thalassemia, congenital adrenal hyperplasia, and Duchenne and Becker muscular dystrophy [135–137]. The disruptive technology of NGS together with the haplotyping strategy is driving the pos-sibility of using NIPD in clinical cases.

PGT has the same diagnostic motivation as the tradi-tional PND, with the advantage of advancing the tim-ing of diagnosis at the embryo stage. Only disease-free embryos are transferred to the mother, avoiding recourse to therapeutic abortion. Even for couples who are able to conceive naturally, PGT requires the application of IVF techniques, including (a) the collection of gametes from both partners; (b) the fertilization of the oocyte by intracytoplasmic sperm injection (ICSI); (c) the embryo biopsy, which allows one or more cells from the blasto-mere or trophectoderm to be taken 3 or 5 days, respec-tively, postfertilization; (d) molecular analysis and (e) the embryo transfer.

PGT protocols are set to start from a small amount of biological sample, ranging from 1 to 10 cells from the embryo at the cleavage stage or blastocyst stage. Conflict-ing opinions are reported on the detrimental effects of embryo biopsy and mosaicism events between cleavage-stage and blastocyst embryos. Linan et al. demonstrated that the concordance of diagnosis in embryos that were double biopsied on D3 and D5 is 67.8% lower than previ-ously reported, supporting the use of blastocyst biopsies instead of cleavage-stage embryo biopsies [138]. Recently, data from the Preimplantation Genetic Diagnosis Inter-national Society (PGDIS) in 2018 showed no difference

in the detrimental effects between the embryo stages if experienced operators performed the biopsies [139, 140].

PGT includes whole genome amplification (WGA) to obtain a sufficient quantity of genomic DNA for one or more molecular investigations [141, 142]. Several types of WGA can be used depending on the downstream applica-tion [142]. The most used technique for PGT is still rep-resented by “multiplex polymerase chain reaction” (PCR) and capillary electrophoresis analysis for the direct iden-tification of the causative mutation of the disease and the analysis of at least two informative polymorphic mark-ers [the most used are the “short tandem repeats” (STR), microsatellites characterized by short tandem nucleotide repeats] or the analysis of at least 3 polymorphic markers in the event that the causative mutation is not known [5,

143]. However, since PGT tailored to a disease is a labori-ous and expensive procedure, which is time-consuming in the preliminary phase for the study of the family, sev-eral laboratories use genome-wide approaches to analyze gene markers throughout the genome. Alan Handyside tested “karyomapping” based on a single nucleotide polymorphism (SNP) array able to determine the geno-type of an individual by analyzing thousands of SNPs distributed throughout the genome [144]. The “karyo-mapping” involves a “linkage” analysis: with a compari-son of the SNPs associated with the causative mutation of the disease to be investigated, that are present in the index case and that are therefore in the parents’ chro-mosomes, to the SNPs present in the embryo cells, it is possible to identify the presence or absence of the muta-tion carrier [144–146]. In addition, the density of the SNPs allows a higher resolution in the case of crossings between chromosomes close to the mutated gene. Finally, it is possible to use “karyomapping” in families with com-binations of more monogenic alterations or that require HLA compatibility, truly demanding investigations to be carried out with conventional methods. “Karyomapping”, however, loses its effectiveness when it is not possible to establish which parental allele is linked to the genetic alteration; for quantitative analysis of mitotic abnormali-ties (mosaicisms), “karyomapping” does not directly ana-lyze the mutation and cannot detect de novo mutations. The aim of most new approaches is to use a targeted SNP analysis to detect single mutations or groups of common mutations combined with quantitative haplotype analysis or chromosome count. Recently, a genome-wide protocol using NGS has been tested for the identification of family mutations together with cytogenetic screening in embryo biopsies [147–149]. The protocol is based on an enlarged panel of disease-associated genes (approximately 5000 genes) and enables, in a single workflow, (a) the direct detection of family mutations and the indirect detection through linkage analysis of heterozygous SNPs (PGT-M);